In 2006, we implemented an HIV and sexually transmitted infection (STI) prevention programme for female sex workers (FSWs) in three Honduran cities. All FSW attending STI clinics underwent regular examinations and STI testing. Information on condom use with different partners was collected at each visit. After three years, we detected a significant decline in the prevalence of syphilis from 2.3% at the first screening to 0.0% at the third screening (P = 0.05), and of chlamydia, from 6.1% to 3.3% (P = 0.01). No changes were observed in the prevalence of gonorrhoea or trichomoniasis. The cumulative HIV prevalence remained constant (P = 0.44). Reports of condom use with clients increased from 93.8% to 98.9% (P < 0.001). The implementation of an HIV/STI prevention programme in FSW has contributed to increases in condom use with clients and the reduction in syphilis and chlamydia prevalence. The intervention should be strengthened and considered as part of a national health policy strategy.