There is little data on the prevalence of STIs in female sex workers, Greek and immigrants, working in Athens, Greece, since most of them work without any form of official license. Our aim was to establish the prevalence of STIs in asymptomatic legal Greek and immigrant female sex workers in Athens, Greece. The study involved an evaluation of gonococcal and chlamydial infection, early infectious syphilis, HIV infection, HSV-2 infection, Hepatitis B and C in 299 female sex workers who applied for an official work permit between May 2005 and October 2005. HSV-2 infection was more common in the Greek sex workers. No difference was found for the other STIs. Prevalence was related to age. A significant association was found between HSV-2 and syphilis. No HIV infection was detected. We concluded that asymptomatic sex workers can be a source of STIs which points out the need for a better health system control in Greece.