Introduction Prevention of Mother-to-Child Transmission of HIV (PMTCT) is a critical component of HIV control and if well implemented drastically reduces transmission to a child. However, Mother-to-Child Transmission of HIV (MTCT) remains a huge burden. Health facility-based antenatal care is still the main vehicle used for PMTCT services in Nigeria, when less than 40% patronize them. In view of shortages of qualified health personnel globally, engaging TBAs will improve the coverage of PMTCT services as it has the potential of bridging the gap. The aim of this study is to provide evidence to inform engagement of TBAs for PMTCT service delivery scale-up in Nigeria. Methods A cross-sectional survey was conducted among TBAs using questionnaires. Total study was done involving 409 TBAs. Data collected was analyzed using IBM SPSS version 25. Chi-square test was used for association of characteristics of TBAs with knowledge and practice on HIV as well as factors influencing willingness to collaborate in the PMTCT programme at < 0.05. Predictors were ascertained using Binary Logistic regression. Ethical Consideration were duly observed. Results In all, 241(58.9%) had overall good knowledge on HIV. The main source of knowledge was Healthcare workers 336 (82.2%). Majority correctly identified ways of HIV transmission and how transmission from infected mother to child can be prevented (>75%). The overall practice on HIV was poor 264 (64.5%) even though the majority 341 (83.4%) refer their HIV clients to health facility. Practices on personal protection was good (>70%). About 378 (92.4%) agree that they have a role in PMTCT for their clients and babies, however only 99 (24.2%) were willing to collaborate in the PMTCT programme. Identified predictors for good practice were; aged ≤ 45 years (AOR 2.66; 95% CI 1.08– 18.46), had primary education and below (AOR 0.68; 95% CI 0.05 – 0.75) and good knowledge (AOR 12.62; 95% CI 8.33 – 44.17). Identified predictors for agreeing to collaborate in the PMTCT programme were good knowledge (AOR 6.34; 95% CI 3.55 – 41.27) and good practice (AOR 3.15; 95% CI 1.74 – 13.35). Conclusions The attainment of PMTCT treatment targets for Nigeria is crucial to that of the world. TBAs are an important but currently underused resource for PMTCT scale-up. There is need to involve TBAs in PMTCT service delivery in Nigeria as if effectively engaged could bridge the gap in HIV control in Nigeria.