The extended prophylactic use of the antiretroviral drug nevirapine in breastfeeding infants of human immunodeficiency virus (HIV)-infected mothers reduces HIV transmission and was associated with a reduction in mortality in HIV-uninfected infants, suggesting possible antimicrobial activity, but there have been no reports of whether nevirapine has antimicrobial activity. To determine whether nevirapine demonstrates antimicrobial activity in vitro, we tested manufactured nevirapine at serum concentrations found physiologically in breastfeeding infants (0.25 g/ml, 0.5 g/ml, 1.0 g/ml, 2.0 g/ml, and 4.0 g/ml) using broth and agar dilution methods. A total of 13 bacteria (comprising eight bacterial species commonly infecting human infants—six ATCC strains and seven patient isolates) were tested. We also confirmed the anti-HIV activity of the nevirapine used in bacterial cultures via qualitative HIV cultures. After testing, MICs against these bacterial organisms exceeded achievable serum concentrations (4 g/ml being the highest concentration tested), and nevirapine showed no activity against any of the organisms tested. In summary, we found no evidence that nevirapine has antimicrobial activity at the concentrations tested against organisms commonly associated with pediatric morbidity and/or mortality.