The role of prenatal antibiotic exposure in the development of childhood wheezing is debated. We evaluated whether this association could potentially be explained by confounding factors.Antibiotic use in the first and third trimester of pregnancy, wheezing in children aged ≤18 months and confounding factors were assessed in singletons participating in the NINFEA (Nascita e Infanzia: gli Effetti dell'Ambiente) birth cohort (n=3530 for first-trimester exposure and n=3985 for third-trimester exposure).There was no evidence of an association between antibiotic exposure in the first trimester of pregnancy and ever-wheezing (adjusted risk ratio (RR) 1.02, 95% CI 0.80-1.30) or recurrent wheezing (RR 0.99, 95% CI 0.54-1.82). For the third-trimester exposure, the crude RRs (95% CI) of ever-wheezing and recurrent wheezing were 1.34 (1.10-1.64) and 2.72 (1.80-4.11), respectively, which decreased to 1.12 (0.90-1.39) and 2.09 (1.32-3.29) after adjustment. The RRs of wheezing after genitourinary infections during pregnancy were increased independently of antibiotic treatment.In conclusion, the association between prenatal antibiotic exposure and infant wheezing could be largely explained by confounding factors, in particular respiratory infections during pregnancy. An excess risk of wheezing after antibiotic exposure during the third trimester of pregnancy remains after adjustment.