Highly seasonal rivers can experience extended low flow, and often dry, periods. Macroinvertebrate and flow data were used to explore hypotheses on the effects of antecedent hydrology and the low-flow, dry-season period on macroinvertebrate assemblages in northern Australia. Composition differed between early and late dry seasons. Taxa were more sensitive to water quality and more rheophilous in the early dry season when their habitats were lotic than when habitats later became lentic. As flow magnitudes in the antecedent dry season and on the sampling day increased, the habitats became more oxygenated and, in turn, macroinvertebrate richness increased. Higher wet-season flow magnitudes, flow variability and rates of fall were correlated with lower richness in the following dry season. Alteration of the flow-disturbance regime that increases the likelihood of flow cessation in macroinvertebrate habitats, or extends the duration of the dry season beyond that previously experienced in these highly seasonal systems, may alter the resistance and resilience of assemblages such that the seasonal decline and recovery of biodiversity may no longer be so reliable. Given the projected increase in low-flow incidence in many regions of the world, future research needs to examine the effects of reduced flow, flow cessation and stream drying as multiple, interacting stressors on stream biota.