Organic matter decomposition is a critical ecosystem process in streams. In urban environments, decomposition is mostly affected by factors associated with altered flow regimes and warm temperatures. Our objective was to assess how urbanization affects organic matter decomposition in a tropical watershed using the cotton strip assay, which reduces variability associated with leaf litter quality and vulnerability to flashy streams. We studied the Río Piedras watershed, a highly urbanized system in the San Juan Metropolitan Area, Puerto Rico. We deployed cotton strips in 7 streams, forming an urban gradient (~10-70% impervious cover). Strips were placed in four pools per stream, collected on two dates depending on the stream identity to ensure that strips reached at least 50% of tensile strength loss. We measured the loss of tensile strength and microbial respiration rates. Environmental variables were pool depth, stream width, pH, conductivity, water velocity, and water temperature. Temperature was measured every hour using HOBO sensors. Water temperature ranged from 26°C in our least urbanized site to 37°C in our most urbanized site. Cotton strip tensile strength loss ranged from ~5-9% per day. There was a positive significant linear relationship between the cotton strips' tensile strength loss and minimum water temperature and significant relationships with watershed land cover variables (i.e. quadratic relationship with impervious surface and tree cover). Microbial respiration rates were high, ranging from 0.38-1.08 mgO2gDM-1hr-1. There was no relationship between the microbial respiration rates and the tensile strength loss of cotton strips. This study shows how urbanization alters organic matter decomposition rates in urban streams. Our results suggest that while tensile strength loss can be explained by the amount of urban development on the watershed, microbial activity may be influenced by other factors. This highlights the complex relationship between urbanization and ecosystem processes.