Freshwater inflows play an important role in delivering dissolved organic carbon (DOC) to estuaries. Although considerable DOC can be delivered to estuaries during episodic inflow events, such as floods, little information exists on how the bioavailability of DOC may change during these periods. In this study we used in vitro bioassay incubation experiments to examine how bioavailability changed following inflow events in two temperate south east Australian estuaries; the agricultural Bega River and the forested Clyde River. We measured short-term (2 days) and long-term (28 days) bioavailable DOC (BDOC) and determined percentage bioavailability, bacterial doubling times (BTd), all with and without excess nitrogen and phosphorus to control for nutrient limitation. Our results showed BDOC varied between 0.13 and 3.62 mg C L−1, equivalent to 2.5–31 % of initial concentrations. BTd were significantly shorter at the peak of flow and reduced as discharge returned to base flow conditions. Multiple-regression analysis showed discharge and specific ultraviolet light absorbance were the best factors for explaining variance in BDOC whilst discharge was the best factor for explaining BTd. The addition of nutrients led to significantly higher measurements of BDOC and bacteria doubling times on the Clyde River when initial phosphorus concentrations were low. These results highlight the importance of freshwater inflow events as intense moments of biogeochemical transformation in estuaries.