The present study indicates the critical role of hydrologic connectivity in floodplain waterholes in the wet-dry tropics of northern Australia. These waterbodies provide dry-season refugia for plants and animals, are a hotspot of productivity, and are a critical part in the subsistence economy of many remote Aboriginal communities. We examined seasonal changes in water quality and aquatic plant cover of floodplain waterholes, and related changes to variation of waterhole depth and visitation by livestock. The waterholes showed declining water quality through the dry season, which was exacerbated by more frequent cattle usage as conditions became progressively drier, which also increased turbidity and nutrient concentrations. Aquatic macrophyte biomass was highest in the early dry season, and declined as the dry season progressed. Remaining macrophytes were flushed out by the first wet-season flows, although they quickly re-establish later during the wet season. Waterholes of greater depth were more resistant to the effects of cattle disturbance, and seasonal flushing of the waterholes with wet-season flooding homogenised the water quality and increased plant cover of previously disparate waterholes. Therefore, maintaining high levels of connectivity between the river and its floodplain is vital for the persistence of these waterholes.