The River Subin, which serves as a source of direct drinking water downstream, flows through the Metropolis of Kumasi in Ghana, making it vulnerable to pollution due to human activities. In this study, we assessed the heavy metals content in the sediment of the Subin River as well as the physicochemical properties and microbial load in the river. Water and sediments were sampled from Race course upstream and downstream, Asafo, and Abinkyi upstream and downstream, Kaase, Asokwa and Wood village with a sampling distance of 100 m. Index of Geoaccumulation (Igeo), contamination factor (CF), contamination degree (CD) and pollution load index (PLI) were used to assess the level of heavy metal enrichment and contamination in the sediments of River Subin. Analysis showed that temperature of the river ranged between 31.23 to 31.40 °C, pH ranged between 6.62 and 7.52, conductivity ranged between 1.16 and 4.67 µS/cm, total hardness ranged from 0.65 to 3.82 mg/L, Ca ranged from 0.26 to 1.22 mg/L, alkalinity ranged from 324 to 711 mg/L, Mg from 0.24 to 3.35 mg/L and Cl ranged from 56.76 to 99. 30 mg/L. The microbial load assessment indicated that the River Subin is highly polluted by E. coli, Faecal Enterococci, Total coliform and faecal coliform, which generally increased as the river flows downstream since more solid waste from domestic and industrial effluents enter the river. Igeo data suggested that the river was highly contaminated by Ag and Cd and moderately polluted by Pb and Co. The CF, CD and PLI, results showed high levels of Ag, Cd, Cr, Cu, Fe, Hg, Co, and Zn, indicating a progressive deterioration of water quality at the various sites. Our results suggest that the River Subin is highly polluted and thus, urgent management strategies are needed to mitigate further deterioration of the river.