Background: Atopic dermatitis (AD) contributes to substantial social and financial costs in public health care systems. Antibiotic exposure during pregnancy has been proposed as a risk factor, but findings remain inconsistent. The aim of this study was to investigate the association between prenatal antibiotic use and childhood AD. Methods: We performed a population-based cohort study using data collected from the Taiwan Maternal and Child Health Database from 2009 to 2016. Associations were determined using Cox proportional hazards model and were adjusted for several potential covariates, including maternal atopic disorders and gestational infections. Subgroup analyses evaluated the influence of postnatal infant antibiotic/acetaminophen use on the association between prenatal antibiotic exposure and childhood AD diagnosed after 1 year of age. Results: A total of 1288343 mother-child pairs were identified and 39.5% received antibiotics prenatally. Maternal antibiotic use during pregnancy was slightly positively associated with childhood AD (aHR 1.05, 95% CI 1.04-1.06), especially in the first and second trimesters. An apparent dose-response pattern was observed with an 11% increased risk when the exposure was ≥5 courses prenatally (aHR 1.11, 95% CI 1.09-1.14). Subgroup analysis showed the positive association remained significant regardless of postnatal antibiotic use; however, a negative association was found in children without postnatal infant acetaminophen use (aHR 1.02, 95% CI 0.97-1.07). Conclusion: Maternal antibiotic use during pregnancy was associated with increased risk of childhood AD in a dose-related manner. Possible confounders existed between prenatal antibiotics and postnatal infant acetaminophen use in the subgroup analysis. Further research may be warranted to investigate this variable using a prospectively designed study, and also to examine whether or not this association is specifically related to pregnancy.