Introduction: As much as 60% of children born in Nigeria are delivered by unskilled traditional birth attendants. It imperative for traditional birth attendants and similar cadre of health care providers in resource-limited settings to be knowledgeable and have the ability to deploy evidence based practices in the prevention of mother to child transmission of HIV if the goal of an AIDS free generation will be achieved. The successful linkage of evidence with practice in sub-Saharan Africa and other resource limited settings will translate into the reduction of MTCT of HIV as has been achieved in other parts of the world. Methods: A cross-sectional survey of 142 traditional birth attendants. The research was based on diffusion of innovation theory. Practices related to prevention of mother to child transmission of HIV were evaluated relative to national guidelines. Linear mixed modelling was used to evaluate the association between PMTCT practices and training on prevention of mother to child transmission of HIV. Results: Most traditional birth attendants were knowledgeable and had good practices relating to prevention of mother to child transmission of HIV. However, significant gaps in HIV exposed infant care; infant feeding practices and harmful traditional practices exist. Conclusions: Traditional birth attendants have a role in the prevention of mother to child transmission of HIV. Evidence-based practices that are related to maternal HIV transmission are being taken from research and policy into patient care domains; though there are some gaps in vital practices. Innovative strategic interventions are very essential to enhance participation of TBAs in the PMTCT of HIV.