Several factors affect periphyton biomass by acting at local and landscape levels simultaneously. Thereunder, we quantified local and land-use variables in a gradient of subtropical streams at the transboundary border between Brazil and Paraguay to determine the influence of some factors (morphology, water dynamics, nutrients, light availability, land use, and number of agrochemicals) on periphyton biomass. We sampled 24 streams (12 in Brazil and 12 in Paraguay) in three different moments (February, July, and November 2019). Biomass was proxied by chlorophyll-a and ash-free dry mass. We also estimated the autotrophic index of periphytic communities as the ratio between ash-free dry mass and chlorophyll-a values. We found that periphyton biomass was structured mainly by stream morphology, water dynamics, and the number of agrochemicals (pesticides) present in the water. Surprisingly, we also found that light availability influenced biomass to a smaller extent, whereas nutrients did not exhibit a significant effect. Our results highlight the influence of stream morphology, water dynamics, and agrochemicals on aquatic biota, demonstrating the importance of conservation of local and regional environments to support the primary productivity and ecosystem functions in streams. [ABSTRACT FROM AUTHOR]