This study was conducted to simultaneously investigate the spatial and temporal dynamics of diatom communities inhabiting two microhabitat types (Sphagnum periphyton and epipelon) in a peatland area of the Czech Republic. The changes in diatom community structure and corresponding diversity indices at both large (i.e., variation between sites) and small (i.e., variation between two microhabitats) scales were assessed through time. The results indicated clear spatial patterns at large, but not at small scale, and only limited seasonal dynamics in the diatom community. At the large scale, significant differences in diatom communities among sites were associated with both geographic position and environmental conditions (pH and conductivity). A significant effect of microhabitat type was detectable within sampling sites; the relationship between other factors and species data was not important on a small scale. The results of this study showed that both diatom diversity and community structure are good indicators of ecological heterogeneity associated with relatively high spatial and/or environmental variability. However, subtle differences in environmental conditions are hardly detectable or hidden using traditional diatom species.