Microphytobenthos play a key role as food resources at the base of the trophic web, particularly in sediments of highly productive seagrass meadows. Here, we investigated patterns of variation in microphytobenthic diatom communities and their driving factors in an impacted versus non-impacted meadow of Ruppia maritima in the Patos lagoon estuary (Brazil), across seasons in two consecutive years. We found higher taxa richness, diversity and equitability, together with lower dominance, at the non-impacted meadow. Seasonally, the same pattern was found in autumn and/or winter 2017 at both meadows, driven by the lower seawater temperature. Higher richness was related to lower river discharge and equitability to lower wind velocity. Diatom assemblages differed in species presence/absence and abundance between meadows, mostly due to higher phosphate at the impacted meadow, together with higher water transparency and sediment organic matter (2016) and with higher grain size (2017) for species presence/absence. Even diatom assemblages differed more in terms of species abundances than of presence/absence, Paralia sulcata and Tryblionela compressa (commonly reported as indicator of eutrophic conditions) were exclusively present at the impacted meadow, while Amphora charrua, Desikaneis howellii, D. simplex (commonly in undisturbed environments) did so at the non-impacted meadow. Often two species were responsible for more than half the dissimilarities in abundances, with Catenula adhaerens dominating at the impacted meadow and Ambo tenuissimus at the non-impacted meadow. Overall, the combination of natural and anthropogenic environmental factors acted together to determine between-meadow and seasonal patterns of variation in the presence and abundance of diatom species.