Sydney Harbour is subjected to persistent stress associated with anthropogenic activity and global climate change, but is particularly subjected to pulse stress events associated with stormwater input during episodic periods of high rainfall. Photosynthetic microbes underpin metazoan diversity within estuarine systems and are therefore important bioindicators of ecosystem health; yet how stormwater input affects their occurrence and distribution in Sydney Harbour remains poorly understood. We utilised molecular tools (16S/18S rRNA and petB genes) to examine how the phytoplankton community structure (both prokaryotes and eukaryotes) within Sydney Harbour varies between high and low rainfall periods. The relative proportion of phytoplankton sequences was more abundant during the high rainfall period, comprising mainly of diatoms, an important functional group supporting increased productivity within estuarine systems, together with cyanobacteria. Increased spatial variability in the phytoplankton community composition was observed, potentially driven by the steepened physico-chemical gradients associated with stormwater inflow. Conversely, during a low rainfall period, the proportion of planktonic photosynthetic microbes was significantly lower and the persistent phytoplankton were predominantly represented by chlorophyte and dinoflagellate sequences, with lower overall diversity. Differences in phytoplankton composition between the high and low rainfall periods were correlated with temperature, salinity, total nitrogen and silicate. These results suggest that increased frequency of high-rainfall events may change the composition, productivity and health of the estuary. Our study begins to populate the knowledge gap in the phytoplankton community structure and substantial changes associated with transient environmental perturbations, an essential step towards unravelling the dynamics of primary production in a highly urbanised estuarine ecosystem in response to climate change and other anthropogenic stressors.