Human activities represent an important cause of degradation to aquatic ecosystems. The replacement of forest cover areas by urbanization can cause changes in ecosystem function as well as the loss of species. In stream ecosystems, these tendencies have been demonstrated for macroinvertebrates and fish. However, there is little information on how the function and diversity of benthic biofilm communities can be affected by land use. The overall objective of this study was to understand the role of watershed and reach factors on the function and diversity of benthic biofilm communities in tropical stream ecosystems. Land use among study streams was analyzed using Global Information Systems (GIS). We measured reach-scale factors like water physicochemistry, canopy cover and photosynthetic active radiation (PAR). Benthic biofilm metabolism was measured as changes in dissolved oxygen concentrations in chambers. Diversity of Bacteria and Eukarya present in biofilms was analyzed using Terminal Restriction Fragment Length Polymorphysm (TRFLP). An experiment was performed to analyze how increases in nutrient concentrations affect benthic biofilm metabolism and diversity. Results indicated that increases in urban land use were significantly related to high nitrogen concentrations (e.g. NO3- and TDN) in streams. Biofilm was primarily autotrophic. Models indicated that metabolism was strongly related to reach factors (e.g. canopy cover and light) rather than land use. Meanwhile, biofilm biomass was greater in urban streams and nitrogen enriched sites. In terms of biofilm diversity, Bacteria and Eukaryotes were related to reach-scale factors (e.g. nutrient concentrations, canopy cover, conductivity, and dissolved oxygen). These results suggested that urbanization can indirectly increase microbial diversity by increasing nutrients in water. Some of the phylotypes identified suggested an anthropogenic effect in benthic microbial vii communities. Results from the nutrient addition experiment supported these results. Higher diversity was found at treatments that contained nitrogen, in comparison to other treatments. However, biofilm metabolism was not affected by the treatments. All these results implied that nutrient additions to streams ecosystems can affect biofilm diversity, and even when changes in metabolism were not a consequence of nutrient additions, changes in ecosystem function cannot be discarded.