Urbanization and agricultural activities are increasingly threatening karstic systems and the water resources they provide, which are crucial for the livelihood of rural communities. Over the past decade, the Río Grande de Comitán-Lagos de Montebello (RGC-LM) watershed in Chiapas, Mexico, has experienced significant deterioration in water quality. The objective of this study was to analyze the water quality dynamics within the RGC-LM watershed and their potential interaction with land use change. We conducted assessments of the physicochemical and microbiological parameters across the lotic and lentic systems within the watershed over eight monitoring campaigns (from 2013 to 2020). Cluster Analysis (CA) and Principal Component Analysis (PCA) statistical approaches were performed to identify the main drivers of surface water quality variability. This approach aims to enhance water quality assessments within environmental management strategies, with the goal of promoting sustainable land-use practices aimed at protecting and improving water resources within the watershed. Our findings indicate that there is a substantial difference between the upper and lower watershed water quality. The good water quality conditions observed in the higher altitude lakes (mountain lakes) can be attributed to favorable topographical features, specific land use patterns, and effective conservation measures. In contrast, the lotic system in the middle watershed and the lentic water bodies located at lower altitudes (plain lakes) exhibit signs of water quality degradation due to inefficient wastewater treatment and agricultural runoff. This study highlights the importance of implementing adequate environmental management strategies to address these water quality challenges effectively. Given the pressing issue of water quality degradation in karstic environments, we emphasize on the need for increased monitoring frequency of both physicochemical and microbiological parameters to capture seasonal variations and to further understand the vulnerability of water resources in karstic environments. Future assessment of nutrient concentrations, pesticide levels, and blue-green algae populations will also be crucial for evaluating the trophic state at a watershed scale.