Diatoms are reliable environmental bioindicators, but their application in coastal environments remains limited. Substrate has been put forward in the literature as an important variable in determining diatom habitat preferences. This study focuses on benthic diatom assemblages and their relationship with substrate specificity in a subarctic tidal environment, which could be relevant for environmental monitoring and management. A variety of substrates were sampled and physicochemical variables measured in various areas of the Bay of Sept-Îles region (northern Gulf of Saint-Lawrence, Canada). We recorded 606 species at 14 sites from 11 substrate types to determine the associations between diatoms and their habitats. Our results suggest that the variability of assemblages in the bay is the result of a combination of the identified variables (temperature, salinity, and total dissolved solids), explaining 26.5% of the variation, and other unmeasured variables (e.g., nutrients, wave action, and currents). Substrate was not identified as a significant variable in the statistical analyses. However, some common species in the surveyed assemblages appeared to show preferences for the substrates they colonized.