Abstract. Estuaries have strong physicochemical gradients that lead to complex variability and often high rates of biogeochemical processes. The biogeochemistry of many estuaries is also increasingly impacted by human activities. Yet our understanding of estuarine biogeochemistry remains skewed towards temperate systems in the northern hemisphere, with far less research from tropical estuaries. This study examined seasonal and spatial variability in dissolved organic matter (DOM) and nutrient biogeochemistry along a partly eutrophic, mixed agricultural/urban estuary system in Southeast Asia, the Johor River and the East Johor Strait. Dissolved organic carbon (DOC) and chromophoric DOM (CDOM) properties showed non-conservative mixing, indicating significant DOM inputs along the estuary. The CDOM spectral slopes and CDOM:DOC ratios suggest that these inputs are dominated by terrigenous, soil-derived DOM along the Johor River, but that phytoplankton production and microbial recycling are more important DOM sources in the Johor Strait. Nitrate consistently showed conservative mixing, while nitrite concentrations peaked at intermediate salinities of 10–25. Ammonia decreased with salinity in the Johor River. In the Johor Strait, however, ammonia increased up to 50 µmol l-1, often dominating the dissolved inorganic nitrogen (DIN) pool. Phosphate was low (<0.5 µmol l-1) throughout the Johor River, but increased in the Johor Strait, where DIN:phosphate ratios were typically at or above 16:1. This suggests that phytoplankton in the Johor Strait may sometimes experience phosphorus limitation. Moreover, internal recycling is likely important for maintaining high nutrient concentrations in the Johor Strait.