Coastal zones are ecosystems that are sensitive to climate change and anthropogenic pollution, resulting in a potential loss of biodiversity and ecosystem services through eutrophication and nutrient imbalances, among others. The coastal El Sauce catchment area, Central Chile, is under multiple anthropogenic pressures including wastewater treatment plant (WWTP) discharge, which its broad effect remains underexplored. In order to assess the impact of the WWTP on El Sauce stream, the benthic microbial communities and key functional groups variability (i.e., nitrifiers, methanogens and methanotrophs) were determined by 16S rDNA high-throughput sequencing and by functional genes quantification, respectively, during two contrasted seasons in three catchment areas (pre-, WWTP and post-discharge). The microbial communities’ structure profiles were associated with the water quality, nutrients, greenhouse gas (GHG) distribution, and the organic matter isotopic signatures in the sediments, for the first time, in this ecosystem. The results show that organic matter isotopic signatures using nitrogen and carbon (δ15N and δ13C) and the physicochemical conditions in El Sauce estuary changed from the pre- to WWTP discharge areas (i.e., a pH decrease of 0.5 units and an increase of 4–6 °C in the water temperature). The WWTP discharge area was characterized by a low nutrient concentration and significantly higher GHG distribution (>600 µM CO2, >30,000 nM CH4, and >3000 nM N2O). In addition, the benthic microbial community structure shifted spatially and seasonally, including specific phyla known as sewage bioindicators, such as Firmicutes (Clostridiales order) and Bacteroidetes. In addition, other taxa were enriched or only retrieved in the sediments of the WWTP influenced area, e.g., Tenericutes, Lentisphaerae, Synergistetes, and LCP-89. Methanogens were more enriched near the WWTP discharge compared to those in the pre-discharge site in both seasons, while methanotrophs and ammonia oxidizers were unfavored only during winter. Our results indicate that the WWTP discharge impacts the biogeochemical conditions in El Sauce catchment area modifying the benthic microbial communities, including a decrease in the key functional groups able to mitigate CH4 and regulate nutrients recycling in these aquatic ecosystems.