Sixty percent of global agricultural production depends on the use of pesticides, despite their adverse effects on human health and the ecosystem. In Mexico, the application of these products has been exacerbated, including pesticides already banned in other countries. The objective of this study was to determine pesticide concentrations in samples of water purification plants and surface water from the Cienega area of Jalisco, Mexico. A survey of 119 farmers with occupational exposure to pesticides was carried out in order to obtain information on the most frequently used pesticides. Subsequently, 51 samples taken at 7 different sites were analyzed using liquid chromatography and mass-mass spectrometry. The most frequently used pesticides were organophosphates (28.87%), pyrethroids (12.89%), and the herbicide paraquat (31.95%). In surface water, the prevalent pesticides were glyphosate (56.96–510.46 ppb) and malathion (311.76–863.49 ppb). Glyphosate levels were higher than the limits acceptable in daily water intake in Cumuato. Malathion levels exceeded the limits permissible by EPA in water purification plants in urban public establishments (100 ppb for children, and 200 ppb for adults). In addition, a multidimensional scaling analysis showed that the sampled sites could be grouped into 2 different bodies of water, based on similarities in their glyphosate concentrations (stress = 0.005), while the concentrations of malathion were heterogeneous (stress = 0.001).