This study provides geochemical partitioning, potential bioavailability, and enrichment of Cd, Cu, Pb, and Zn in bottom sediments collected from the Matanza-Riachuelo River and its main tributary streams. A modified Tessier sequential extraction procedure, complemented with acid volatile sulfide (AVS) and simultaneous extracted metals (SEM) measurements, was applied to determine the partitioning of metals into four fractions (metals bound to amorphous sulfide, carbonate, and exchangeable), bound to Fe/Mn oxides (reducible), bound to organic matter/sulfide (oxidizable) and residual. Spatial and vertical distributions of metals were studied. The core sediments show a decreasing concentration of metals with depth. In top sediments, non-residual Cu was mainly associated with oxidizable phase, whereas Pb, Cd, and Zn were mainly associated with amorphous sulfide. Pb exhibited the highest enrichment in all sites. The ratio AVS/SEM was greater than one at sediment sections close to the water column, indicating that metals extracted with hydrochloric acid were mainly associated with the amorphous sulfide. The strong influence of amorphous sulfide in the retention of Cd, Pb, and Zn in anoxic sediments of Matanza-Riachuelo river system suggests that dredging and aeration could lead to the remobilization of metals from sediments to the water column, hence making the metals more available to the biota.