Background and objective <p>The role of the microbiome and gut flora alterations in childhood obesity has drawn increasing scientific attention. However, large-scale, long-term cohort studies with real-world data on exposure and outcomes are lacking. We aimed to examine the association between exposure to antibiotics during infancy and the development of overweight and obesity during childhood and adolescence.</p> Methods <p>We conducted a historical cohort study using data from a large Israeli health provider (Maccabi Healthcare Services, MHS). Eligible patients born between 1998-2002 who received antibiotics before age 2 years were compared with unexposed infants. Valid body mass index (BMI) data were available for 76,840 eligible infants, including 65280 exposed to antibiotics (52.2% males) and 11,560 unexposed (46.1% males). Study outcomes were overweight and obesity during childhood/ adolescence.</p> Results <p>Compared to unexposed, antibiotic-exposed infants had significantly higher mean ( ± SE) BMI percentiles in all age groups: childhood (57.8 ± 0.1 vs 55.0 ± 0.2), early adolescence (58.2 ± 0.1 vs. 55.1 ± 0.3), and late adolescence (57.4 ± 0.2 vs. 55.0 ± 0.4), all <i>p</i> &lt; 0.001. The odds of overweight and obesity versus unexposed infants increased significantly (<i>p</i> &lt; 0.001) with the number of dispensed narrow-spectrum antibiotic packs, from an odds ratio of 1.15 (95%CI: 1.02–1.29) for 1–2 packs, to 1.52 (95%CI: 1.13–2.05) among those exposed to 10 or more packs. No such association was found for broad-spectrum antibiotics.</p> Conclusions <p>Exposure to narrow spectrum antibiotics during infancy was associated with a higher BMI and an increased likelihood of overweight and obesity in childhood and adolescence.</p>