Discharge is a primary determinant of river phytoplankton, but its influence can be mediated by climate, water quality and catchment development. The relationship between discharge and phytoplankton over seasonal and flood temporal scales was examined for the Daly River in the Australian tropical savannah. Phytoplankton, water quality and hydrographic data were collected over the high-discharge wet season and lower-discharge dry season. Wet season main channel river conditions were unfavourable for phytoplankton growth. Floods, however, connected the main channel to off-channel water bodies that supplied phytoplankton, predominately Cryptomonas, to the river. Wet season phytoplankton biovolume and cell concentrations were higher than in the dry season and comprised the majority of the annual phytoplankton biomass load. High discharges served to both temporally connect the river to off-channel phytoplankton sources as well as dilute these sources. In the dry season, the Daly River was groundwater-fed and disconnected from off-channel phytoplankton sources and dominated by a potamoplanktonic population of Cryptomonas and Peridinium. River phytoplankton were determined by the seasonal discharge regime which drove water quality and underpinned a shift from highly productive, spatially extensive off-channel sources in the wet season to less productive more spatially confined in-channel sources in the dry season.