Abstract. Estuaries have strong physicochemical gradients that lead to complex variability and often high rates of biogeochemical processes, and they are also often impacted by humans. Yet, our understanding of estuarine biogeochemistry remains skewed towards temperate latitudes. We examined seasonal and spatial variability in dissolved organic matter (DOM) and nutrients along a partly eutrophic, agricultural–urban estuary system in Southeast Asia: the Johor River and the East Johor Strait. Dissolved organic carbon (DOC) and coloured DOM (CDOM) showed non-conservative mixing, indicating significant DOM inputs along the estuary. The CDOM spectral slopes and CDOM : DOC ratios suggest that terrigenous, soil-derived DOM dominates along the Johor River, while phytoplankton production and microbial recycling are important DOM sources in the Johor Strait. CDOM properties were not unambiguous source indicators in the eutrophic Johor Strait, which is likely due to heterotrophic CDOM production. Nitrate concentrations showed conservative mixing, while nitrite concentrations peaked at intermediate salinities of 10–25. Ammonium concentrations decreased with salinity in the Johor River but increased up to 50 µmol L−1 in the Johor Strait, often dominating the dissolved inorganic nitrogen (DIN) pool. Phosphate concentrations were low (<0.5 µmol L−1) throughout the Johor River but increased in the Johor Strait, where DIN : phosphate ratios were typically ≥ 16 : 1. This suggests that the Johor Strait may experience phosphorus limitation and that internal recycling is likely important for maintaining high nutrient concentrations in the Johor Strait. Overall, our results indicate that the Johor River and Johor Strait are clearly not part of the same estuarine mixing continuum and that nutrient recycling processes must be quantified to understand nutrient dynamics in the Johor Strait. Moreover, our results highlight the need for better techniques for DOM source tracing in eutrophic estuaries.