Cenotes are spectacular karst formations in Yucatan, Mexico, often used for recreation. However, their impact on water quality has yet to be explored in detail. Therefore, during Easter, water samples were collected from four cenotes to identify variations in water quality associated with the presence of tourists. PCO of water quality, before (PH) and during Holy Week (HW) in 2019, explained 49.02% of the total variation. The indicators contributing to the first principal coordinate’s variation were Sr, K, sulfate, and chloride (0.89). Whereas, alkalinity, temperature, conductivity, nitrate, and ORP contributed to the second PC. PERMANOVA indicated a significant interaction between “cenote” and “condition” factors, and post hoc paired comparisons indicated significant differences between PH and HW conditions. Significant correlations varied among the four cenotes as the result of hydrogeological differences. Whereas, numbers of visitors were correlated with at least one fecal-matter indicator, demonstrating anthropogenic influence on the cenotes’ water quality.