In this study we evaluated the magnitude and seasonal variations of natural and anthropogenic fluxes of inorganic (NO3−, NH4+, and PO43−) and organic (DON and dissolved organic carbon) nutrients delivered by submarine groundwater discharge (SGD) and rivers to the fourth largest estuary in the USA, Mobile Bay in Alabama. To identify the sources of SGD-nutrient in the estuary and their subsurface biogeochemical transformation, we applied a multi-method approach that combines geochemical nutrient (N and P) mass-balances, stable isotopes (nitrate $$\updelta^{15} {\text{N}}_{{{\text{NO}}_{3} }}$$ and $$\updelta^{18} {\text{O}}_{{{\text{NO}}_{3} }}$$ and sediment organic matter δ13Corg and δ15Norg) signatures, microbial sequencing analyses, dissolved organic matter source-composition, and shallow estuarine sediment lithological analyses. We found that during dry seasons SGD delivered nearly a quarter of the total nutrient inputs to Mobile Bay. These SGD fluxes were anoxic and N was delivered to the bay almost entirely as NH4+ and DON, which represented more than half of the total NH4+ and almost one fifth of the total DON inputs to the bay. We further observed that these significant SGD-derived N fluxes occurred exclusively to the east shore of Mobile Bay, historically impacted by hypoxia and large-scale fish kills known as “Jubilees”. We demonstrate here that although the Mobile Bay coastal area is largely developed and anthropogenic influences are well documented, a shallow peat layer identified only on the east shore serves as the main source of the exceptionally high NH4+ and DON fluxes. We found that the high groundwater NO3− concentrations observed further inland from over-fertilization also identified by previous studies, decreased dramatically as groundwater percolated through the intertidal zone of the coastal aquifer. The microbial community identified in the coastal sediments suggests that denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were the main processes responsible for this extensive removal and transformation of anthropogenic N, respectively. Furthermore, we found no significant anthropogenic inputs from manure or sewage waste to the bay. These findings show that natural sources of nutrients can outcompete anthropogenic inputs despite extensive development of the coastal area. We hypothesize that similar subsurface biogeochemical nutrient transformations can occur in other shallow estuaries of the northern Gulf of Mexico and worldwide.