Understanding the impacts of tidal barriers on macrobenthos is essential for comprehending anthropogenic influences on coastal ecosystems and biodiversity. This study aimed to address two primary questions: firstly, whether there are differences in macrobenthos abundance and diversity between the protected (CPA) and non-protected areas (CNPA) characterized by the tidal barrier, and secondly, whether the environmental factors associated with tidal barriers influence the macrobenthos community structure. Samples collected from CPA and CNPA sites from a subtropical river-estuary revealed the presence of 16 taxa of soft-bottom invertebrates, with a predominant presence of Ampeliscidae, Capitellidae, Glyceridae, Oligochaeta, Mysidae, Mytilidae, Naticidae, Portunidae, and Insecta. Significant differences (p < 0.05) in salinity and conductivity were observed between CPA and CNPA. In the CPA area, the average temperature was 23.21°C, dissolved oxygen was 4.81 mg/L, and pH was 7.91, all of which were higher compared to CNPA.Macrobenthos abundance was higher in CNPA (1506 ± 688 ind.m−2) compared to CPA (1330 ± 611 ind.m−2) (p < 0.05) with distinct dominance patterns of Polychaeta and Malacostraca. CNPA showed lower species dominance index (0.10–0.24), higher species diversity index (1.52–2.38), slightly higher Margalef`s index (from 0.63 to 1.6), and lower Shannon Index (from 0.69 to 0.85) indicating a more balanced community structure with a more diverse assemblage of species than CPA and a moderately polluted area. Hierarchical cluster analysis (HCA), Non-metric multidimensional scaling (nMDS) analysis, and Canonical Correspondence Analysis (CCA) indicated that the tidal barrier negatively impacted the macrobenthic families. The findings indicated the influencing effect of the breakwater defensive system on the macrobenthos assemblage at the estuary and informed policymakers and environmental scientists to develop environment-friendly breakwater defense systems.