Background: Infantile exposure to macrolides has been associated with hypertrophic pyloric stenosis causing projectile vomiting, dehydration, electrolyte abnormalities, and in rare cases death possibly via macrolide interaction with gastric motilin receptors. Large population-based cohorts have suggested that exposure to macrolides via breastmilk may be associated with pyloric stenosis. Methods: In this prospective, controlled observational study designed to assess the safety of macrolides during lactation, we followed infants whose mothers contacted our Drug Consultation Center at the Assaf Harofeh Medical Center (Zerrifin, Israel) inquiring about safety of macrolides during lactation and compared them to a cohort of infants exposed to amoxicillin during breastfeeding. Results: Fifty-five infants exposed to macrolide antibiotics were compared to a control cohort of 36 infants exposed to amoxicillin via lactation. The infants in the macrolide group were all exposed to erythromycin and the newer macrolides: azithromycin, clarithromycin, and roxithromycin. The rate of adverse reactions the infant experienced while being exposed to both antibiotics was comparable. Seven (12.7%) infants in the macrolide group experienced adverse reactions versus three infants (8.3%) in the amoxicillin group (odds ratio = 1.6, 95% confidence interval, 0.38-6.65, p = 0.73). The adverse reactions in the infants exposed to macrolides were rash, diarrhea, loss of appetite, and somnolence, whereas the infants exposed to amoxicillin experienced rashes and somnolence. Factors such as gestational age, age and weight at exposure, maternal age, or type of macrolide were not associated with the infant's adverse reaction in multivariate regression analysis. Conclusions: Rates and types of minor adverse reactions in breastfed infants exposed to a macrolide or amoxicillin in breastmilk were comparable. Macrolide exposure during breastfeeding was not associated with pyloric stenosis, although larger prospective studies are required to confirm our observation.