The chemical species of metals (Fe, Ni and Pb) in the Middleton River were determined and their relationship with soil organic matter was established with a view to identifying the likely source (natural or anthropogenic) of these metals within the water body. The GBC Avanta PM AAS was used to quantify the levels of metals in each fraction. The most important fractions of iron, nickel and lead were found to be the poorly mobile and immobile metal species. The metals were most affiliated to the residual fractions thereby depicting that they are from the natural environment. However, the dry season depicted the tendency for nickel and lead to become bioavailable and potentially toxic as significant levels of the metals were superficially adsorbed on the surface of sediment colloids where they are readily redistributed within the water column as water soluble fractions. Sediment organic matter in the Middleton River may be mainly composed of soluble fulvic acids which may have aided metal desorption. The order of prevalence for metal speciation in the dry season was organic matter bound (24.7%), residual (67.6%) and organic matter bound fraction (34.3%) for iron, nickel and lead respectively. The wet season revealed the prevalence of amorphous Fe-oxide (28.6%), residual fraction (78.0%) and organic matter bound fraction (30.4%) for iron, nickel and lead respectively, thereby, portending that the metals are emanating from the natural environment. On the other hand, the readily transportable water soluble fractions depicted significant metal retention levels of (19.1%) and (12.5%) for nickel and lead during the dry season thereby portending the contribution of anthropogenic inputs; most likely from activities of oil bunkers, occasional oil spill from worn-out pipeline joints and boat transportation. Metal mobility factors depicted the trends (Ni>Pb>Fe) for the impacted areas across the dry and wet seasons. The control locations also depicted (Pb>Ni>Fe) and (Ni>Fe>Pb) during the dry and wet seasons respectively. Overall, Ni and Pb may possess the most potential to be bioavailable in the Middleton River estuary.