A professor of pathological anatomy (1844–74) at the Vienna General Hospital, he inspired the Bohemian student Ignaz Semmelweis (Semmelweis, Ignaz Philipp), later a martyr to the cause of antiseptic medical practice, to take up the study of medicine (1846) and afterward supported him in his struggle to eliminate childbed fever by cleaning up Europe's maternity wards.

First to detect bacteria in lesions of malignant endocarditis, an often rapidly fatal inflammation of the membrane lining the inner walls of the heart, Rokitansky created the basis for a differentiation of lobar pneumonia (originating in the lower lobe of the lung) and lobular pneumonia, or bronchopneumonia (originating in the finer subdivisions of the branched bronchial tree). He made a fundamental study of acute yellow atrophy of the liver (now known as Rokitansky's disease; 1843), established the micropathology of pulmonary emphysema (a condition of the lung characterized by enlarged air spaces separated from the terminals of the bronchial tree), and first described spondylolisthesis (1839), the forward displacement of one vertebra over another.

His Handbuch der pathologischen Anatomie, 3 vol. (1842–46; Treatise of Pathological Anatomy, 1849–52), represented an elevation of the discipline to the status of an established science.