In aquatic systems worldwide, heavy metal pollution has been increasing alongside rapidly growing anthropogenic activities, and most heavy metals are stored in sediments. Overlying water conditions may influence whether sediments act as heavy metal sinks or sources. In this study, we investigated the effects of the dissolved oxygen (DO) and nutrient levels of overlying water on the total contents and fractions of Pb, Zn, Ni, Cu, Mo, and Fe in river surface sediments. Sediments and overlying water were collected from a tributary of the Hai River in Tianjin, China, and then incubated for 61 days under laboratory conditions. The chemical speciation of heavy metals was determined following the modified Community Bureau of Reference (BCR) three-step sequential extraction procedure. The results showed that Pb, Zn, and Fe were released from the sediments in an anoxic environment and adsorbed from the overlying water in an aerobic environment. High nutrient levels facilitated the adsorption of Pb, Zn, Cu, and Fe in the sediments, while the total content of Mo was higher under low nutrient level conditions. The DO and nutrient levels appeared to have no influence on the total content of Ni. According to the risk assessment code classification (RAC), anoxic conditions decrease the potential bioavailability risks of Pb, Zn, Ni, Cu, Mo, and Fe. Anoxic conditions can also reduce the percentage of the potentially mobile fractions of Pb, Zn, Cu, and Fe. The low nutrient level contributed, to some extent, to reducing the potential bioavailability risk of Pb, but increasing the risk of Cu. The high nutrient level increased the potentially mobile fractions of Pb, Zn, Cu, and Fe. The information obtained in this study improves our scientific understanding of the effects of overlying water conditions on the total heavy metal contents and fractions.