AbstractA major goal of microbial ecology is to establish the importance of spatial and environmental factors in driving community variation. Their relative importance likely varies across spatial scales, but focus has primarily been on free-living communities within well-connected aquatic environments rather than less connected island-like habitats such as estuaries, and key host-associated communities within these systems. Here we sampled both free-living (seawater and sediment) and host-associated (estuarine fish hindgut microbiome,Pelates sexlineatus) communities across six temperate Australian estuaries spanning ∼500 km. We find that spatial and environmental factors have different influences on these communities, with seawater demonstrating strong distance-decay relationships (R= −0.69) and significant associations with a range of environmental variables. Distance-decay relationships were weak for sediment communities but became stronger over smaller spatial scales (within estuaries,R= −0.5), potentially reflecting environmental filtering across biogeochemical gradients or stochastic processes within estuary sediments. Finally,P. sexlineatushindgut microbiome communities displayed weak distance-decay relationships (R= −0.36), and limited variation explained by environmental variables, indicating the significance of host-related factors in driving community variation. Our findings provide important ecological insights into the spatial distributions and driving forces of both free-living and host-associated bacterial patterns across temperate estuarine systems.