Groundwater represents almost half of the drinking water worldwide and more than one third of water used for irrigation. Agro-industrial activities affect water resources in several manners; one of the most important is leaching of agrochemical residues. This research identifies the major contributors of changes in groundwater quality comparing two contrasting land uses in a karstic area of the Yucatan peninsula as case study. Using a multiple approach, we assess the impact of land use with physicochemical data, multivariate analyses, hydrogeochemistry and nitrate isotopic composition. We confirmed that agricultural land use has a greater impact on groundwater quality, observed in higher concentration of nitrates, ammonium, potassium and electrical conductivity. Seasonality has an influence on phosphates and the chemical composition of the groundwater, increasing the concentration of dissolved substances in the rainy season. There was a clear effect of manure application in the agricultural zone and the nitrate isotopic composition of groundwater points toward recharge in certain areas. We consider that seasonality and land use effects are intertwined and sometimes difficult to separate, likely because of land use intensity and hydrogeochemical process at a local scale. Finally, we observed poor groundwater quality in the agricultural area during the wet season; thus, it is desirable to maintain non-agricultural areas that provide groundwater of appropriate quality.