Vertical transmission of HIV is responsible for about 14 % of new HIV cases reported each year in sub-Saharan Africa. Barriers that prevent women from accessing and using antiretroviral medications (ARVs) for themselves and their infants perpetuate the epidemic. To identify influences on access to and use of infant HIV health services, specifically nevirapine administration, we conducted a mixed methods study among HIV-positive women in Uganda. This included a cross-sectional survey (n = 384) and focus group discussions (n = 6, 5-9 participants each). Of the 384 women, 80 % gave nevirapine to their infants within 72 h of birth. Factors independently associated with nevirapine administration were lack of maternal adherence to ARVs (AOR 3.55, 95 % CI 1.36-9.26) and attending a support group (AOR 2.50, 95 % CI 1.06-5.83). Non-health facility births were inversely related to nevirapine use (AOR 0.02, 95 % CI 0.003-0.09). Focus group discussions identified four themes impacting access and use: attending a support group, health care worker attitudes, lack of partner support, and poor health messaging regarding ARVs. Improving health care worker messaging regarding ARVs and providing women with needed support to access and use infant ARV prophylaxis is critical to overcoming access barriers. Eliminating these barriers may prevent numerous HIV infections each year saving the lives of many HIV-exposed infants.