This study describes HIV, sexually transmitted infections (STI) and risk factors associated with Mycoplasma genitalium among female sex workers (FSWs) in four cities in Honduras. In 2006, 795 FSWs from Tegucigalpa, San Pedro Sula, La Ceiba and Comayagua were recruited using respondent-driven sampling (RDS) and tested for HIV prevalence and STI. HIV prevalence ranged from no infections in Comayagua to 5.4% in Tegucigalpa. With the exception of Comayagua, more than 20% of FSWs were infected with M. genitalium. M. genitalium in the aggregated cities was associated with HIV positivity, being aged ≤30 years old, drinking alcohol more than once weekly and always using condoms with regular clients in the past month. In comparison with a 2001 surveillance study we found lower rates of HIV infection. Interventions for HIV control and prevention among FSWs, including promotion of condom use, are needed in Honduras.