We assessed the potential effects of urbanization on bioavailability of dissolved organic C (DOC) in stream ecosystems, by quantifying bacterial extracellular enzymatic activity (EEA). We measured activities of 6 enzymes by incubating water samples with 4-methylumbelliferyl substrates from streams across an urbanization gradient east of Melbourne, Australia. A principal components analysis (PCA) ordination separated streams according to their relative urbanization, as indicated by effective imperviousness (proportion of each catchment covered by impervious surface directly connected to streams by pipes). Activities of leucine aminopeptidase and esterase were higher in streams in more urbanized catchments than in less-urbanized streams, where moderate activities of a diverse range of enzymes were observed. High relative contributions of leucine aminopeptidase and esterase to overall enzyme activity in urban streams stressed the increased importance of peptides as a C source for heterotrophic bacteria and nonspecific extracellular hydrolytic activity. In contrast, high contributions of β-N-acetyl glucosaminidase and β-xylosidase in less-urbanized streams highlighted the significance of microbial detrital material as a C source and processing of plant-derived xylooligosaccharide substrates in these environments. Our results suggest a shift in organic C bioavailability across streams of contrasting urbanization, despite all streams having roughly similar DOC concentrations. We propose that relative EEA rates show promise as an ecological indicator of stream health across an urban gradient.