The response of planktonic bacteria and phytoplankton to various additions of dissolved organic carbon (DOC) as glucose, with and without inorganic nutrients (nitrogen and phosphorus), was tested in the upper to mid Hunter Estuary, Australia. In situ microcosms (1.25 L) were performed at two sites with varying salinities over three seasons. Analysis of variance showed a significant difference among control and treatments for all seasons for the bacterial, dissolved oxygen and chlorophyll a responses (P < 0.05). A significant interaction between treatment and site was found in autumn for dissolved oxygen, autumn and spring for bacterial and spring for chlorophyll a responses. At both sites for each season, and on nearly all occasions, bacterial surface area was enhanced by DOC addition as indicated by both increased bacterial abundance and dissolved oxygen utilisation. DOC in combination with inorganic nutrients sometimes further enhanced the bacterial response compared to DOC alone. Inorganic nutrients alone did not enhance growth of the heterotrophic bacterioplankton. Addition of DOC alone led to decreased chlorophyll a relative to the control, probably due to competition for limited inorganic nutrients with the bacterioplankton DOC non-limiting conditions. Results suggest that the heterotrophic community was limited by DOC at both sites and across seasons. An experiment with a larger volume (70 L), performed over a longer time, compared a control with DOC addition. Increased bacterial biomass as a result of DOC addition occurred at day 2. Chlorophyll a did not significantly differ between treatments. An increase in zooplankton density was recorded in the DOC treatment relative to the control at day 10. This study supports the contention that increased DOC delivery with river inflows through environmental flow allocations will stimulate heterotrophic bacterioplankton production in the upper Hunter Estuary.