Port sites represent one of the most impacted coastal areas; this impact is due to intensive anthropogenic pressures. In addition to the port complex itself, associated activities, such as indiscriminate disposal of pollutants, including trace metals, affect the local ecosystem. Macroinvertebrate benthic communities are one of the most effective bioindicators of environmental health because of their importance as a primary food source for many fish, birds, and mammals, as well as their influence on sediment stability and geochemical composition. This article evaluates the benthic macrofauna in the Santos Estuarine System (SES), the location of the Santos Port Complex (SPC), linking trace metal levels to differences in microbenthic community structure and pollutant bioavailability. The distribution of Cd, Ni, and Pb was directly related to organic matter deposits, while Cu and Zn appeared to result from port activities. The SES contained a poor benthic macroinvertebrate community, resulting from the contaminated muddy sediments. A significant negative correlation was found between the macrobenthic diversity and concentrations of Cu in the soluble phase; this implied the pollution-induced degradation of the macrobenthos in SES.