Nigeria is second in the world for the number of people with HIV and has a high rate of mother-to-child transmission (MTCT). Over 60% of births in Nigeria occur outside of health care facilities, and because of this, Traditional Birth Attendants (TBAs) play a significant role in maternal and child health. It is important that TBAs be knowledgeable about HIV prevention. The purpose of this study was to determine the impact of HIV testing and counseling (HTC) knowledge on the HIV prevention practices among TBAs in Nigeria. Five hundred TBAs were surveyed. Chi-square and logistic regression were used to assess differences in HIV prevention practices between TBAs with and without HTC knowledge. TBAs with HTC knowledge are significantly more likely to engage in HIV prevention practices than TBAs without HTC. Prevention practices included: wearing gloves during delivery (p < 0.01), sterilization of delivery equipment (p < 0.01), participation in blood safety training (p < 0.01), and disposal of sharps (p < 0.01). As long as a high percent of births occur outside health care facilities in Nigeria, there will be a need for TBAs. Providing TBAs with HTC training increases HIV prevention practices and can be a key to improve maternal and child health.