Background: Prevention of mother to child transmission of HIV plays a major role in reducing the number of children being infected with HIV. This study provides baseline information about the knowledge, practice and factors that affect PMTCT service provision by traditional birth attendants. Methods: A cross-sectional study of 142 traditional birth attendants in Ogun State, Nigeria carried out between January and March, 2014. Sample selection was by two-staged probability sampling technique. Data collection employed structured pre-tested questionnaires. Data were analysed using SPSS software for Windows (version 18). Uni-variate, bi-variate and multivariate analyses were done. Results: Participants were universally aware of HIV. Participantsâ€™ ability to correctly identify temporal mode of MTCT of HIV varied from 69.0% for ante-partum; and 76.1% during labour and delivery to 60.6% during breastfeeding. Most of them (81.7%) knew that MTCT of HIV is preventable. Most of them knew that knowledge of HIV status (94.4%); and HCT (98.6%) were important for pregnant women. The mean score for knowledge of MTCT and PMTCT of HIV was 16.89 Â± 1.89. Participants with secondary education or more (AOR = 3.020, 95%, CI = 1.362-6.697) were likely to know more about MTCT and PMTCT of HIV. Majority (88.7%) of the TBAs always offer HCT to pregnant women; 122 (85.9%) always refer HIV-positive women to treatment centres; and 126 (88.7%) always provide referral support for patients. The mean score for PMTCT care practice for the participants was high (45.85 Â± 9.48). Conclusion: TBAs have a role to play in the e-MTCT of HIV in Nigeria and are poised to do so. They have some knowledge and good practices as it relates to MTCT and PMTCT of HIV. Some misconceptions and bad practices exist especially as it relates to infant feeding practices. These need to be comprehensively addressed.