The bioavailability and composition of dissolved organic carbon (DOC) and nitrogen (DON) were examined in 10 major sub-catchments of the Swan-Canning estuary, which bisects the city of Perth, in south-western Australia. Catchments contain a mix of forest, agriculture, and urban-dominated land-use, with the degree of development increasing near the city center. We incubated water samples from the 10 sub-catchments for 14 days at 25°C, and measured changes in DOC and DON and dissolved inorganic nitrogen (DIN). A greater proportion of DON (4–44%) was decomposed compared to DOC (1–17%). Both agricultural and urban catchments had high proportions of bioavailable DOC and DON, but overall DOC and DON losses were greatest in urban catchments. Using resin isolation techniques, we found that DOC was concentrated in the hydrophobic (humic) fraction, whereas DON had both hydrophobic and hydrophilic (non-humic) fractions. Hydrophobic DOC content was positively related to DOC decomposition. In contrast, DON decomposition was highly correlated with hydrophilic DON content and inversely related to the hydrophilic DOC/DON ratio, indicating a labile fraction of DON from non-humic sources. Taken together, these relationships suggest that bioavailable DOC may be supplied in part from terrestrial plant material, but bioavailable DON is likely to be from highly labile sources, possibly autochthonous or anthropogenic. Overall, labile DON was greater than initial DIN concentration at seven of ten sites and was even dominant in highly developed catchments. This study highlights the importance of organic N in urbanizing coastal catchments that, in addition to DIN, may serve as a readily available source of N for in-stream and estuarine production.