Environmental DNA (eDNA) metabarcoding provides a powerful, non-invasive approach for assessing biodiversity in aquatic ecosystems. This study investigates spatiotemporal variation in community composition across eleven estuaries in northern New South Wales, Australia, including three within the Cape Byron and Solitary Islands Marine Parks. Sampling captured seasonal dynamics and disturbance associated with flooding from ex-tropical Cyclone Alfred, enabling assessment of ecosystem resilience and recovery trajectories. Surface water samples were collected across upper, middle, and lower estuarine zones in three intensively sampled sentinel estuaries and eight reduced-effort surveillance estuaries. Metabarcoding targeted whole-community assemblages, allowing comparison of biodiversity patterns and evaluation of microbial communities as bioindicators of ecosystem condition. Presence–absence data were analysed using multivariate approaches to detect shifts in species richness, community composition, and drivers including rainfall, land use, and catchment characteristics. Significant shifts in community composition were observed following the cyclone, with concordant responses across microbial and whole ecosystem assemblages, and partial recovery evident within two months. Land use influenced recovery trajectories, while sustained rainfall appeared to constrain resilience in some systems. These findings improve understanding of estuarine responses to extreme flood events and support scalable, cost-effective eDNA-based biomonitoring for coastal management under increasing climate variability.