Urbanization is increasing worldwide, but drivers and the ultimate fate of dissolved organic matter (DOM) are uncertain. Urban waters are often characterized by high algal and microbial productivities that transform allochthonous and autochthonous sources of DOM. We analyzed dissolved organic carbon (DOC) concentrations, DOM composition, and bioavailable DOM (BDOM) within man-made urban canals of Miami, FL (USA). We sampled surface water from 17 sites during wet (July 2021; September 2021; June 2022) and dry (January 2022; April 2022) seasons. Aquatic metabolism (gross primary productivity, GPP; ecosystem respiration, ER) was measured using light/dark bottle changes in dissolved oxygen, and microbial extracellular enzyme activities related to carbon, phosphorus, and nitrogen acquisition were measured. BDOM was quantified as changes in DOC concentrations and DOM composition over 28-day lab incubations. Results showed seasonal changes in BDOM, with microbial activity influencing DOM composition during the wet season, not the dry season. DOC concentrations remained stable throughout incubations, and terrestrial humic-like DOM was prevalent across the landscape, with significantly higher levels in the dry season (p < 0.001). During the wet season, humic DOM levels decreased in incubations (p = 0.026), indicating microbial degradation of recalcitrant DOM. Aquatic metabolism was net heterotrophic (GPP<ER), although no relationship was observed between metabolism and enzyme activities or BDOM. In wet-season incubations, carbon-acquiring enzymes were strongly correlated with decreasing humic DOM and DOC concentrations. Conversely, in dry-season incubations, increases in labile, microbially-derived DOM were correlated with nitrogen-acquiring enzymes. Our findings reveal that seasonal variability in DOM composition is driven by microbial transformations related to seasonal bioavailability and demand of dissolved carbon and nutrients.