The present study documents the first systematic assessment of a brothel in Bangladesh in terms of sexually transmitted disease (STD) and human immunodeficiency virus (HIV). A cross sectional study was undertaken on brothel-based commercial sex workers (CSWs) selected systematic random sampling to assess the prevalence of STDs and HIV among CSWs in a brothel setting. Two hundred and ninety-six CSWs were selected from a brothel with a population of 593 women. Following informed consent, endocervical and blood samples were obtained for the diagnosis of genital chlamydia, gonorrhoea, HIV and syphilis respectively. In addition, another 170 consecutive blood samples were collected from the total CSW population for HIV tests. All blood samples for HIV testing were made anonymous by removing patient identifiers before testing. Endocervical specimens were tested by polymerase chain reaction (PCR) for the diagnosis of genital chlamydia and gonorrhoea. Syphilis and HIV infections were diagnosed by serology. One hundred and sixty-nine (57.1%) of the women were Treponema pallidum haemagglutination (TPHA)-positive, 20 (6.8%) of the women were Venereal Disease Research Laboratory (VDRL)-positive at a greater than 1:8 dilution. Eighty-two (28%) of the women were found to be infected either by gonorrhoea or chlamydia. No HIV antibody was found in any of the 466 blood samples. A high prevalence of STDs and low prevalence of HIV in the CSWs in Bangladesh suggest potential for the rapid spread of HIV once it is introduced in this high-risk population. The opportunity to control STD and HIV infection in this population should not be missed, in order to prevent a large epidemic in the future.