BackgroundYellow fever is an acute febrile illness caused by a mosquito-borne flavivirus. The discovery that mosquitos were responsible for transmission and that the disease was preventable by vector control, as well as the development of an efficacious vaccine (in the 1930s) have reduced its public health impact. However, underutilization of vaccination and discontinuation of vector control measures have led to yellow fever outbreaks, affecting thousands of people in Africa and South America, and is a continued threat to people who travel to endemic regions without vaccination. An additional concern is that the vaccine has important contraindications, and the currently available doses are insufficient to immunize the populations under risk of contracting yellow fever. Specific antiviral chemotherapy would be an alternative to vaccination, however there is none yet available. Drug repurposing is one strategy that can speed up drug discovery and development and has been underexplored for yellow fever. Methods A high content screening assay for the discovery of anti-yellow fever compounds was developed and used to screen a library of pharmacologically compounds, which includes approved drugs and covers most signalling pathways and all major drug target classes. The same library was screened against cells infected with a serotype 2 of the dengue virus. Results and conclusionFrom 1280 compounds screened, 88 compounds (6,9% of the library) were found to reduce yellow fever virus (YFV) infection in 50% or more. Interestingly, the number of compounds that presented similar activity against dengue (DENV) infection was considerably lower (18 compounds, 1,4%). Among these, 12 compounds were active against both viruses, highlighting the potential of finding compounds with broad antiviral activity in diversity libraries. The top 27 anti-YFV compounds were selected for further activity determination in dose-response. Five compounds (Gant61, benztropine mesylate, brequinar sodium salt hydrate, PF-429242 dihydrochloride and U-73343) presented selective activity against YFV infection in a human cell line, but only two of them, brequinar and U-73343, were also active against DENV. These compounds, which represent a broad spectrum of pharmacological functions, have not been previously described as having anti-YFV activity, thus offering a valuable opportunity for the development of specific antiviral chemotherapy for yellow fever.