Various studies report contrasting results on the substrate-type effect on diatom community composition, but the particularly important question is whether or not it affects diatom-based assessments of water quality. We investigated whether the substrate type is a significant predictor of the diatom community composition and if it affects lake water quality assessment based on diatom indices. This study took place in Sava Lake (Serbia). We used glass, ceramic, willow and yew tree tiles as artificial substrates for periphyton development, and pebbles from the lake littoral as natural substrate. Results revealed differences in both the diatom community composition and diatom indices values related to the substrates. A distinction was recognized between natural, artificial wooden, and artificial inert substrates. However, the final lake quality assessment based on diatom indices was more or less similar in all substrate types in our study, and depended on value ranges associated with water quality classification and on diatom index choices. Artificial substrates in our study did show potential as an alternative for natural substrate, but further studies are required, particularly in various types of lentic ecosystems to confirm our findings and support artificial substrate employment in lake water quality assessment.