Landscape urbanization broadly alters watersheds and stream ecosystems, yet the impact of nonpoint source urban inputs on the quantity, quality, and ultimate fate of dissolved organic matter (DOM) is poorly understood. We assessed DOM quality and microbial bioavailability in eight first-order Coastal Plain headwater streams along a gradient of urbanization (i.e., percent watershed impervious cover); none of the streams had point source discharges. DOM quality was measured using fluorescence excitation-emission matrices (EEMs) coupled with parallel factor analysis (PARAFAC). Bioavailability was assessed using biodegradable dissolved organic carbon (BDOC) incubations. Results showed that watershed impervious cover was significantly related to stream DOM composition: increasing impervious cover was associated with decreased amounts of natural humic-like DOM and enriched amounts of anthropogenic fulvic acid-like and protein-like DOM. Microbial bioavailability of DOM was greater in urbanized streams during spring and summer, and was related to decreasing proportions of humic-like DOM and increasing proportions of protein-like DOM. Increased bioavailability was associated with elevated extracellular enzyme activity of the initial microbial community supplied to samples during BDOC incubations. These findings indicate that changes in stream DOM quality due to watershed urbanization may impact stream ecosystem metabolism and ultimately the fate of organic carbon transported through fluvial systems.