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The aim of the present study was to assess the exposure to pesticides in urine from Spanish lactating mothers (n = 116). Six nonspecific (dialkyl phosphates) and 20 specific metabolites of organophosphate pesticides (OPs), herbicides and pyrethroids were analyzed. The most frequently detected biomarkers were diethyl phosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinol and 3-phenoxybenzoic acid, whose geometric means were 1.9 ng·mL−1, 0.8 ng·mL−1, 1.5 ng·mL−1 and 1.4 ng·mL−1, respectively. Herbicide metabolites were the least frequently detected biomarkers with detection frequencies between 0% (2,4,5-Trichlorophenoxyacetic acid) and 22% (2,4-Dichlorophenoxyacetic acid). Multiple regression analyses showed that the closeness to a farming activity, the place of residence and the presence of garden/plants at home were some of the most important contributors to urinary levels of pesticide metabolites. Estimated daily intake (EDI), hazard quotient (HQ) and hazard index (HI) were obtained in order to interpret urinary levels of the most frequently detected pesticide metabolites in a risk assessment context. The highest EDIs were obtained for chlorpyrifos (0.40–1.14 μg·kg bw−1·day−1) and deltamethrin (0.34–4.73 μg·kg bw−1·day−1). The calculated HQ for chlorpyrifos, dimethoate, parathion and deltamethrin ranged from 0.01 to 0.47, and HI for OPs ranged from 0.09 to 0.33 showing that apparently there were low health risks due to the exposure to these pesticides in this group of Spanish breastfeeding women. Unlabelled Image • 26 pesticide biomarkers were analyzed in 116 urine samples from lactating women. • The highest detection frequency (91%) and GM (1.9 μg/L) were obtained for DEP. • Place of residence and the use of pesticides are major predictors of OP exposure. • Spanish mothers were more exposed to pyrethroids than those from other countries. • Risk assessment for OPs and pyrethroids showed hazard quotients lower than 1. [ABSTRACT FROM AUTHOR]