This paper estimates population-based prevalence of HIV, syphilis, HSV-2 and factors influencing HIV infection using a national sample of 1914 female sex workers (FSWs) in 7 regions in Tanzania. Additionally, HIV incidence was estimated by comparing biological HIV results with self-reported HIV status. The average HIV prevalence among FSWs in all 7 regions was 28%, ranging from 14% in Tabora to 38% in Shinyanga. HIV incidence was found to be 13 per 100 person-years. Syphilis prevalence was 8% with significantly higher burden found in Iringa (11%), Mbeya (13%), and Shinyanga (12%). Nearly 60% of the study population was infected with HSV-2. The high HIV prevalence and incidence coupled with suboptimal condom use indicate an urgent need to roll out the “Treat-All” approach and provide antiretroviral therapy to FSWs living with HIV regardless of their CD4 count. In addition, antiretroviral-based prevention technologies such as oral pre-exposure prophylaxis and microbicides should be piloted and evaluated.