The Fusarium toxin zearalenone (ZEN) is of concern due to its pronounced estrogenic effects in mammalian species. ZEN contaminates various cereal crops and grain-based food along with modified forms which contribute to overall mycoestrogen exposure. As no data exist on the occurrence of ZEN in food commodities consumed in Bangladesh, we have analyzed ZEN and its main metabolites α-and β-zearalenol (α-ZEL, β-ZEL) by targeted LC-MS/MS method as biomarkers of exposure in urines (n = 62) from rural and urban residents in Rajshahi district collected in two seasons and from a pregnant women cohort (n = 20) in Dhaka district. Average levels of α-ZEL, the far more potent estrogenic metabolite, were clearly higher than those of ZEN and β-ZEL. Biomarker levels in urban and rural residents showed some seasonal fluctuation: In winter urines, ZEN mean level was 0.040 ± 0.037, α-ZEL 0.182 ± 0.047 and β-ZEL 0.018 ± 0.016 ng/mL; in summer urines, ZEN mean was 0.028 ± 0.015, α-ZEL 0.198 ± 0.025 and β-ZEL 0.013 ± 0.005 ng/mL. In pregnant women, mean levels were: ZEN 0.057 ± 0.041, α-ZEL 0.151 ± 0.026 and β-ZEL 0.055 ± 0.057 ng/mL, thus similar to levels found in the Rajshahi cohort in winter season. Estimates of probable dietary mycoestrogen intake in the Bangladeshi adults reveal an exposure below the tolerable daily intake of 0.25 μg/kg b.w. set by EFSA.