Diatoms are good indicators of water quality in lotic systems. The purpose of this study was to provide information about the effects of natural and artificial substrates on diatom-based water quality assessment. Two artificial substrates (bricks and glass) were placed at 6 sampling sites and left for a month during summer base-flow period (2008). Water quality variables were measured at the beginning and end of the experiment. The IndVal method was used to find indicator species and species assemblages characterizing the three substrates. Species richness, diversity and equitability differed among sampling sites, tending to be higher in relatively unpolluted compared to polluted sites. The relatively less polluted upstream sites were characterized by such species as Aulacoseira ambigua, Aulacoseira granulata, Cymbopleura naviculiformis, Eunotia bilunaris, Fragilaria capucina and Gomphonema angustatum. On the other hand, the highly polluted downstream sites were characterized by Gomphonema parvulum, Nitzschia palea, Pinnularia amazonica and Synedra ulna. Species diversity and richness differed between the substrates, tending to be high on natural compared artificial substrate. Indicator species analysis showed that common diatom species were not restricted to single substrate. However, some species tended to prefer certain substrates as suggested by their highest indicator values in these preferred substrates. Specificity was generally high for natural compared to artificial substrates. Among the artificial substrates, more species tended to prefer glasses compared to bricks. Pollution tolerant species, N. palea, G. parvulum and Achnanthidium minutissimum, were highly associated with artificial substrates. Substrate differences may affect the interpretation of water quality results because the absence of a particular species on a given site is can be mistaken for the effects of the perturbations under study. The use of natural substrate is recommended compared to artificial substrate given the advantages of the former compared to the latter.