Benthic biofilm metabolism in streams can be strongly affected by watershed land use. As riparian vegetation cover decreases, a shift in metabolism occurs towards high primary production. We measured biofilm metabolism in nine low-order streams in a watershed under increasing development pressure in Puerto Rico using closed sealed metabolic chambers. Three streams were selected within the three major land use types in the watershed: forest and low intensity agriculture, mix agriculture and rural communities, and suburban land uses. Percent of land use was determinate using GIS tools. We hypothesized that the main impact of land use was related to increases in light availability with a change from heterotrophic to autotrophic metabolism. Results supported this hypothesis, showing that streams with riparian vegetation had low nutrient levels and high respiration rates. In contrast, those in mix agriculture and suburban land uses had high nutrient levels and high rates of primary production. Overall, our study shows that benthic biofilm metabolism reflects the land use type on the watershed and that rates are mainly related to changes in riparian vegetation cover and in light availability. Assessment of biofilm metabolism could be a useful tool in understanding anthropogenic impacts to lotic ecosystem function.