Benthic algae are a major source of carbon supporting aquatic food webs in northern Australia, but little is known about the factors that regulate algal production. We surveyed benthic algal biomass in mainstem habitats of an unregulated sandy tropical river (the Fitzroy River) during a base-flow period. We used predictive models to reveal the physical and chemical parameters controlling algal biomass in mainstem habitats. We found that water velocity was an important driver—algal biomass was lower at higher water velocities. Subsurface flow was also influential—algal biomass increased in locations where groundwater upwelling occurred, as evident by a positive relationship between algal biomass and elevated radon and ammonium concentrations. In this sand-bed river, it is likely that high water velocity destabilises the sandy substrate reducing the establishment of algal biofilms. However, where water velocity is low enough for algal establishment, groundwater upwelling likely promotes algal growth by delivering limiting resources and/or creating stable conditions that promote algal production. The importance of surface and subsurface-flow conditions to benthic algal biomass means that any modification to the Fitzroy River catchment that alters dry-season longitudinal flows (via river regulation) or groundwater levels (via groundwater extraction) may directly influence river algal production.