Many streams of southwestern Australia have become secondarily saline through land clearance and other human activities in their catchments. Elevated salinities impact on aquatic biota and ecological processes of surface streams but little is known of the effects on the diversity and community composition of hyporheic (subsurface) invertebrates occupying the saturated sediments where surface and groundwaters exchange. We hypothesized that biodiversity of hyporheic invertebrates would decline with increasing salinity, especially where saline groundwater upwelled into the surface stream. We also predicted changes in community composition associated with salinity and direction of vertical hydrological exchange. Water and hyporheic invertebrates were sampled from downwelling and upwelling zones of 13 streams in southwestern Australia ranging in median surface water salinity from 0.27 to 17.86 g L⁻¹. Overall, taxa richness of hyporheic invertebrates was uncorrelated with salinity but, surprisingly, correlated positively with the salinity of upwelling water. However, when the sites were divided into 'fresh' (<3 g L⁻¹) and 'mesosaline' (>3 g L⁻¹) groups, this relationship became non-significant. Instead, taxa richness and total abundance were correlated positively with salinity of downwelling water in fresh sites and negatively in mesosaline sites, resulting in a peak in richness at intermediate salinities. Community composition was unrelated to direction of hydrological exchange but was strongly associated with hyporheic salinity. Hyporheic assemblages of 'fresh' rivers were typified by harpacticoid copepods and candoniid ostracods, whereas the amphipod 'Austrochiltonia' and several dipteran groups were more common below 'mesosaline' rivers. Although many hyporheic taxa collected in this study apparently have broad tolerances to salinity, secondary salinization due to human activities potentially changed community composition, possibly altering rates of ecological processes such as organic matter breakdown occurring within the sediments of streams undergoing salinization.