The aim of this study was to determine the effect of prevention of the mother to child transmission (PMTCT) program in Benin City, Nigeria on the prevalence of postnatal infant HIV. Effects of the duration of PMTCT, place of birth and sex of the infants on the prevalence of postnatal HIV were also assessed. Dried blood spots were collected from 318 infants (6–8 weeks old) born to HIV-positive mothers and were screened for the presence of HIV using a qualitative polymerase chain reaction method. A questionnaire was used to obtain information about the duration of highly active antiretroviral therapy (HAART) and place of delivery. Male infants had a significantly (p = 0.032) higher prevalence of postnatal HIV infection than females. Nonparticipation of mothers in the PMTCT program was a significant risk factor for acquiring postnatal HIV (odds ratio = 4.519; 95% confidence interval = 2.422, 8.429; p < 0.0001). The duration of HAART use significantly (p = 0.010) affected the prevalence of postnatal HIV with an inverse relationship. The place of delivery had no effect on the prevalence of postnatal HIV. An overall prevalence of 16.98% of postnatal HIV was observed in this study. Male sex and no participation in the PMTCT program were significant risk factors for acquiring postnatal HIV, while a lower prevalence of postnatal HIV infection was associated with longer use of HAART in the PMTCT program. We recommend an early diagnosis of maternal HIV status and commencement of HAART.