This observational study was designed to determine the prevalence of HIV infection and the microbial isolates from the genital tracts of couples attending the fertility clinics of Olabisi Onabanjo University Teaching Hospital, Sagamu, Ogun State, Nigeria between January 2001 and December 2002. A total of 110 women and 49 of their male partners were recruited into the study. The majority of the patients were between 25 and 34 years (women: 71.8%; partners: 65.3%), though the men were significantly older than the women (p < 0.001). The overall prevalence of HIV infection in those who consented to screening was 8.2%, which was over twice the reported prevalence among the general population in Ogun State, Nigeria during the study period. Candida albicans (32.5%) and Staphylococcus aureus (27.5%) were the most frequently isolated microorganisms from the endocervix while Trichomonas vaginalis (37.9%) and Staphylococcus aureus (24.1%) were the most common microbes isolated from the posterior vaginal fornix. Of all the seminal qualities, only the volume showed a significant difference between the infected and non-infected samples (p < 0.004). This study suggests a higher prevalence of HIV infection among the infertile couples in our environment and it may be advisable to have them screened for HIV in the face of the present HIV situation in sub-Saharan Africa.