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 one year using a grab Ekman sampler with a sampling area of 0.3 m² in three replicates. Collected samples were fixed with 4% formalin and transferred to the Gilan University laboratory. Over the one-year sampling period, a total of 745 macrobenthic samples were isolated and identified. The greatest taxonomic diversity was observed among bivalves, particularly in the families Mytilidae and Cardiidae. Among the identified macrobenthos, the invasive species Amphibalanus improvisus was recorded in the river estuary. The detection of this species in a brackish and riverine environment indicates its high adaptability and habitat expansion potential, which could pose a warning for the structure of benthic communities. Some physicochemical 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 macrobenthic 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 invasive species.