Benthic algae are a major source of carbon supporting aquatic food webs in tropical northern Australia, but little is known about the factors that regulate algal production particularly over small scales. We surveyed benthic algal biomass in mainstem habitats in an unregulated sand-bed tropical river during a base-flow period. Physicochemical parameters (e.g. water velocity, substrate, water quality) were measured at each sampling point and groundwater upwelling (as indicated by radon) and nutrients were measured at a sub-set of points. We used predictive models to reveal the factors controlling algal biomass in mainstem habitats. We found that water velocity was an important driver - algal biomass was lower at higher water velocities. Sub-surface flow was also influential - algal biomass increased in locations where upwelling occurred, as evident by a positive relationship between algal biomass and elevated radon and ammonium concentrations. Micro-algal constituents (diatoms, green algae, cyanobacteria) displayed the same pattern as total algal biomass. In this sand-bed river, it is likely that high flow velocity destabilises the sandy substrate preventing the establishment of algal biofilms. However, where flow velocity is low enough for algal establishment, groundwater upwelling promotes algal growth by delivering limiting resources (e.g. nutrients) and/or creating stable physicochemical conditions that promote algal production. The importance of surface and sub-surface flow conditions to benthic algae biomass means that any modification to the Fitzroy River catchment that alters dry-season longitudinal flows (via river regulation) and/or groundwater levels (via groundwater extraction) may directly influence river algal production.