This study aimed to investigate the seasonal diversity of macrobenthic communities in the substrate of the Shalmanrud River estuary and to identify ecologically important species in the area. Following preliminary field surveys, four stations along a 600-meter stretch from the estuary to the upstream section were selected, and monthly sampling was conducted over a period of one year using a Ekman grab sampler having a sampling area of 0.3 m² and done in three replicates. Collected samples were fixed in 4% formaldehyde fixative and transferred to the laboratory of the Faculty of Basic Sciences, University of Guilan, for further research. Over one-year of sampling, a total of 745 macro benthic specimens were isolated and identified. The greatest taxonomic diversity was observed among bivalves, particularly in the families Mytilidae and Cardiidae. Among the identified macro benthos, the alien species Amphibalanus improvisus was also recorded. The detection of this species in a brackish and riverine environment indicated its high adaptability and habitat expansion potential, which could pose a warning for the structure of benthic communities. Some physico-chemical parameters, including water temperature, ambient temperature, pH, depth, and transparency, were also measured. Canonical Correspondence Analysis (CCA) revealed that water temperature and transparency were the most influential factors affecting the spatial distribution of dominant families, especially Mytilidae and Cardiidae, across different stations. Biodiversity was evaluated using Shannon, Simpson, and Pielou’s indices with the aid of PAST software. The study demonstrates that macro benthic diversity indices are a suitable tool for ecological monitoring, assessing the biological health of estuary-influenced rivers, and detecting biological threats such as the presence of alien species.