This historical review examined the onset of the vaccination method during the Ottoman Empire. Inoculation was performed in the regions of Thessaly, Macedonia, and Thrace using folk medicine as a measure against the spread of smallpox/variola infection. Greek physicians Emmanuel Timonis (1669–1720) and Jacobus Pylarinos (1659–1718) as well as several other Ottoman scientists of the Greek or Turkish descent pioneered the use and dissemination of variolation and the development of vaccination before or concurrently with Edward Jenner (1749–1823). During the 19th century in the Adrianople (Edirne) region and much earlier in Constantinople (İstanbul), vaccination programs used to be implemented as evidenced by various certificates distributed at that time. Ottoman vaccination documents from the early 20th century and the letter of Lady Mary Wortley Montagu (1689–1762), dated 1719, have been analyzed, which confirms the extensive use of the vaccination method. Smallpox was the first disease to have been treated with vaccination method. The difference between the Greek and Ottoman physicians and Edward Jenner lies in the fact that while the Greek and Ottoman physicians removed fluid from pustules of an infected person to perform inoculation, Edward Jenner removed fluid from pustules of infected cows, which is why Edward Jenner’s method was coined vaccination (derived from the Latin word “vacca” meaning “cow”). Further, Turkish physicians Mustafa Behçet Efendi (1774–1834) and Sanizade Mehmed Ataullah Efendi (1771–1826) recommended the variolation method. It thus appears that the Ottomans provided care to all ethnicities of their Empire. Vaccines were initially used against smallpox, but the immunization program was eventually extended to other diseases.