Billroth was born at Bergen auf Rügen in the Kingdom of Prussia, the son of a pastor. His father died of tuberculosis when he was five years old. He attended school in Greifswald where he obtained his Abitur degree in 1848. Billroth was an indifferent student, and spent more time practicing piano than studying. Torn between a career as a musician or as a physician, he acceded to his mother's wishes and enrolled himself at the University of Greifswald to study Medicine, but gave up the whole of his first term to the study of music; Professor Wilhelm Baum, however, took him with him to Göttingen, and his medical career was fixed. He then followed Professor Baum to the University of Göttingen, and completed his medical doctorate at the Frederick william University of Berlin in 1852. Along with Rudolph Wagner (1805–1864) and Georg Meissner (1829–1905), Billroth went to Trieste to study the torpedo fish.
From 1853 to 1860 Billroth was an assistant in Bernhard von Langenbeck’s surgical clinic at the Charité in Berlin. There he was also apprenticed to Carl Langenbuch. In 1860, Billroth accepted an offer from the University of Zurich to become the Chair of Clinical Surgery, becoming Director of the surgical hospital and clinic in Zurich. The beginning of his career in Switzerland was unpromising: during his first semester of teaching, he had only ten students, and he himself said that the income he received from his private practice was insufficient to pay for his morning cup of coffee. His reputation quickly grew however; Billroth had an infectious personality, attracting both students and surgical trainees to his ranks. He was loved by his students, and was an effective undergraduate as well as graduate Teacher. Students flocked to his lectures, and with the cooperation of energetic colleagues, he was able to raise the Medical Faculty of Zurich to a prominent position among German speaking schools in only a few years.
Billroth was a talented amateur Pianist and Violinist. He met Brahms in the 1860s, when the Composer was a rising star of the Viennese musical scene. They became close friends and shared musical insights. Brahms frequently sent Billroth his original manuscripts in order to get his opinion before publication, and Billroth participated as a musician in trial rehearsals of many of Brahms' chamber works before their first performances. Brahms dedicated his first two string quartets, Opus 51, to Billroth.
While in Zurich, Billroth published his classic textbook Die allgemeine chirurgische Pathologie und Therapie (General Surgical Pathology and Therapy) (1863). At the same time he introduced the concept of audits, publishing all results, good and bad, which automatically resulted in honest discussion on morbidity, mortality, and techniques – with resultant improvements in patient selection.
He was appointed professor of surgery at the University of Vienna in 1867, in succession to Franz Schuh; there, he practiced surgery as chief of the Second Surgical Clinic at the Allgemeine Krankenhaus (Vienna General Hospital). Though he laid the foundation of his fame at Zurich, it was in Vienna, a larger and more conspicuous theater, that he established himself as the power that he was in the surgical world.
He did not limit himself to surgery only, and conducted extensive research on an ailment that affected many surgery patients at the time: wound fever. His treatise on wound fever, Untersuchungen über die Vegetationsformen von Coccobacteria septica (1874; “Investigations of the Vegetal Forms of Coccobacteria septica”) concluded that the cause was bacterial; Billroth was quick to use antiseptic techniques in his surgical practice, and the number of surgical patients afflicted with wound fever greatly decreased. With the threat of fatal surgical infections lessened through his work and others’, Billroth proceeded to turn his attention to surgery and the pioneering field of altering or removing organs that had previously been considered inaccessible.
An early adopter of the "white coat" (as shown in Seligmann's c.1890 painting), Billroth was directly responsible for a number of landmarks in surgery; in 1872, he was the first to conduct an esophagectomy, removing a section of the oesophagus and joining the remaining parts together. In 1873, he performed the first laryngectomy, completely excising a larynx. He was the first surgeon to excise a rectal cancer and by 1876, he had performed 33 such operations. By 1881, Billroth had made intestinal surgery seem almost commonplace. But his most famous accomplishment is unquestionably the first successful gastrectomy for gastric cancer. On January 29, 1881, after many ill-fated attempts, Billroth performed the first successful resection for antral carcinoma on Therese Heller, who lived for almost 4 months and died of liver metastases. He accomplished this operation by closing the greater curvature side of the stomach and anatomizing the lesser curvature to the duodenum, in an operation that is still known as the Billroth I to this day.
Billroth's literary activity was widespread, with the total number of published books and papers of which he was the author numbering about one hundred and forty. He collaborated, with von Pitha in a Textbook of General and Special Surgery (1882). To this, Billroth contributed the section on Scrofulosis and Tuberculosis, Injuries and Diseases of the Breast, Instruments and Operations, Burns, Frostbites, etc.
In 1887 Billroth was made a member of the Austrian Herrenhaus, "House of Lords"; a distinction rarely bestowed on members of the medical profession.
Billroth and Brahms, together with the acerbic and influential Viennese music critic Eduard Hanslick, formed the core of the musical conservatives who opposed the innovations of Richard Wagner and Franz Liszt. In the conflict, known as the War of the Romantics, Billroth supported Brahms, but was always fair and measured in his comments. "Wagner was indeed a very considerable talent in many directions," he wrote in 1888.
During the Franco-Prussian War, Billroth did excellent work in the military hospital at Mannheim and Weissenburg, treating a variety of horrific battlefield injuries with aggressive and ambitious surgeries; he embodied his experience of war surgery in his Surgical Letters from Mannheim and Weissenburg. He was so impressed by the horrors of war, that he was ever afterwards an ardent advocate of peace. On December 3, 1891, he delivered an address on the care of the wounded in war which made a profound sensation and led to large sums of money being voted by the Austrian legislative chambers for the provision of adequate means of succour for the wounded.