Transfer of trace metals into fish of commercial value poses a public health concern. Therefore, sediments, invertebrates, and fish from three sampling sites in the Rufiji estuary were analyzed for trace metals to evaluate their concentrations and trophic transfer within estuarine food web. Stable isotopes of carbon and nitrogen were also used to study the trophic relationship between different organisms. Biomagnification of trace metals in organisms from different trophic levels was quantified and evaluated by calculating the bioaccumulation factor and biomagnification factor. Trophic magnification factor for different trace metals was determined from the slope of the regression line between trace metal concentration and the trophic level of functional groups in sampled organisms. The results indicated that As and Zn displayed trophic level-dependent accumulation in the Rufiji food webs. As and Zn increased with the trophic level, whereas Ag, As, Co, Cr, Cu, Mn, Ni, and Pb depicted an opposite trend. Food web magnification factors varied from -0.57 for Ni to 0.39 for Zn, whereas trophic magnification factor varied from 0.27 for Ni to 2.47 for Zn. Zn and Ni bioaccumulate in the food webs as indicated by a slope greater than zero, whereas the remaining trace metals are eliminated from food webs or their trophic transfer is interrupted.