The potential use of epilithic diatoms as indicators of organic pollution was evaluated in Gravataí River, RS, (latitude 29°45′–30°12′ S; longitude 50°27′–51°12′ W). The river suffers agricultural impacts in its upper course and urban and industrial organic pollution in its lower course. Epilithic diatoms were sampled eight times from September 2000 to August 2002, at six sites. Species were identified and densities and relative abundances of populations were determined. Simultaneously, physical, chemical and microbiological variables were measured (water temperature, conductivity, turbidity, pH, dissolved oxygen, biochemical oxygen demand (BOD5), chemical oxygen demand, ammonium, organic nitrogen, total nitrogen, ortho-phosphate, total phosphate, chloride and faecal coliforms). In order to interpret the environmental and biological variables, discriminant analysis and the TWINSPAN methods (Two-Way Indicator Species Analysis) were applied. The results indicated that the concentrations of ortho-phosphate, ammonium, total organic nitrogen, BOD5 and faecal coliforms characterized a pollution gradient along the river, where changes in the abundance or species composition were observed. Species were classified into three groups: Group A, including species more tolerant to heavy organic pollution and eutrophication, represented by Luticola goeppertiana, L. mutica, Eolimna subminuscula, Nitzschia palea and Sellaphora pupula; Group B, comprised of tolerant and widely distributed species such as Eunotia bilunaris, Frustulia crassinervia, F. saxonica, Navicula cryptocephala, N. cryptotenella, Nitzschia palea var. tenuirostris, Surirella angusta, Pinnularia microstauron and Ulnaria ulna and Group C, with less pollution tolerant species represented by Eunotia sp. and Gomphonema parvulum.