Aquatic environments face a variety of threats in the Hondo River basin, Southeastern Yucatán Peninsula. Some of these impacts, like pollution by pesticides, may depend on land use and cover. Our objective was to assess the effect of land use/cover using a previously published index of biotic integrity (IBI), based on fishes and designed for shallow streams in the Hondo River basin. Our hypothesis was that land uses that cause deforestation and pollution, such as urbanization, cattle breeding, or sugar cane fields, would be reflected in low IBI values, at least at some spatial scales. The 23 sites originally used in [2008][2009] to estimate by electrofishing the relative abundance and other characteristics of selected fish species and guilds to construct the IBI, were revisited in February 2010, to validate by direct inspection the type of land use/cover suggested by landscape information in digital databases. We analyzed the effect of seven types of land use/cover (agriculture, pasture, human settlements, water bodies, wetlands, forest, and secondary vegetation) on the IBI values, at four spatial scales, i.e., the percent of every land use/cover at progressively larger circles (125, 250, 500, and 1 000 m diameter) centered on the water body where the IBI value was measured. Correlations were established among the percent land/use cover by scale around 23 sites, and with their corresponding IBI values. Then, Student's t tests were calculated to examine significant differences in land use/cover between groups of localities above and below the median IBI value, and Mann-Whitney's U tests were applied to compare IBI values between localities with and without a given landscape cover. Agriculture, human settlements, and secondary vegetation correlated negatively with the IBI; forests positively. Differences were significant (p<0.05) for forest (higher in sites with higher IBI values) and human settlements (lower in sites with higher IBI). Of all the landscape categories located in the Hondo River basin, with the exception of pasture, those of anthropogenic origin tended to be detrimental to aquatic biotic integrity. Rev. Biol. Trop. 65 (4): 1448-1458. Epub 2017 December 01.Key words: human impact, fishes, environmental risk, landscape dynamics, index of biotic integrity, aquatic diversity.Along the Mexican-Belizean Hondo River basin, most human settlements used to subsist on forestry, agriculture, livestock, and tourism (although since the early 2000s most of them rely on government subsidies and remittances from the United States or from Cancún and Playa del Carmen: Castellanos, 2010). Some of these activities threaten freshwater systems due to creation of waste, such as organic matter and pesticides, among others. Agriculture, transport, and urban residual water are the main sources of pollution (Buenfil-Rojas, Álvarez-Legorreta, & Cedeño-Vázquez, 2014;Sánchez, Álvarez-Legorreta, Pacheco, Carrillo, & González, 2016). Additionally, the presence of invasive species in the region, like tilapia Oreochromis mo...