Worldwide, it is acknowledged that changes of land use influence water quality; however, in tropical forests, the relationship between land use and water quality is still poorly understood. This study assessed spatial and seasonal variations in water quality, and the relationship between water quality and changes of land use in the Bobos-Nautla River, whose upper course runs across a patch of a tropical cloud forest. Spatial and seasonal variations in water quality and land use were assessed with multivariate tools. A cluster analysis, as well as a Principal Component Analysis (PCA-3D), identified three groups of sites: (1) an upper portion, which showed the best water quality and the broadest natural vegetation coverage; (2) a middle course, with high nitrogen and phosphorus concentrations associated with extensive agricultural uses; and (3) a lower course, characterized by the highest levels of total and fecal coliforms, as well as ammonia nitrogen, associated with the highest percentage of urbanization and human settlements. Our findings demonstrate the impact of changes of land use on water quality of rivers running through cloud forests in tropical zones, which are currently endangered ecosystems.