Aflatoxin B1 (AFB1) is the most potent of the dietary aflatoxins, and its major metabolite, aflatoxin M1 (AFM1), is frequently found in the breast milk of lactating mothers. The aim of this study was to assess the occurrence and factors associated with AFM1 contamination of breast milk collected from lactating mothers in Lebanon. A total of 111 breast milk samples were collected according to the guidelines set by the World Health Organization. Samples were analyzed with a competitive enzyme-linked immunosorbent assay between December 2015 and November 2016. A survey was used to determine the demographic and anthropometric characteristics of participating lactating mothers. Dietary habits were assessed using a semiquantitative food frequency questionnaire. Mean (±standard deviation) concentration of AFM1 in the breast milk samples was 4.31 ± 1.8 ng/L, and 93.8% of samples contained AFM1 at 0.2 to 7.9 ng/L. The mean concentration of AFM1 was significantly lower (P < 0.05) in fall and winter (4.1 ± 1.9 ng/L) than in spring and summer (5.0 ± 1.7 ng/L). None of the samples exceeded the European Commission regulation limit (25 ng/L) for infant milk replacement formula. AFM1 contamination was significantly associated (P < 0.05) with the daily consumption of white cheeses but not with the consumption of meat or cereal products. No significant association (P > 0.05) was observed between AFM1 concentrations in breast milk and anthropometric sociodemographic factors (age and level of education) or the governorate of residence of the nursing mothers. The mean AFM1 estimated daily intake was found to be 0.69 ng/day/kg of body weight. Although the incidence of AFM1 contamination was low, our first-of-its-kind study highlights the importance of conducting investigations on mycotoxin contamination in breast milk and of developing protection strategies to tackle the exposure of infants to this potent chemical hazard.