The Maumee River watershed in the Laurentian Great Lakes Basin has been impacted by decades of pollution and habitat modification due to human settlement and development. As such, the lower 35 km of the Maumee River and several smaller adjacent watersheds comprising over 2000 km² were designated the Maumee Area of Concern (AOC) under the revised Great Lakes Water Quality Agreement in 1987. As part of pre-rehabilitation assessments in the Maumee AOC, we assessed fish and invertebrate communities in river km 24–11 of the Maumee River to identify: 1) areas that exhibit the highest biodiversity, 2) habitat characteristics associated with high biodiversity areas, 3) areas in need of protection from further degradation, and 4) areas that could feasibly be rehabilitated to increase biodiversity. Based on benthic trawl data, shallow water habitats surrounding large island complexes had the highest fish diversity and catch per unit effort (CPUE). Electrofishing displayed similar fish diversity and CPUE patterns across habitat types early in the study but yielded no discernable fish diversity or CPUE patterns towards the end of our study. Although highly variable among study sites, macroinvertebrate density was greatest in shallow water habitats <2.5 m and around large island complexes. Our results provide valuable baseline data that could act as a foundation for developing rehabilitation strategies in the lower Maumee River and for assessing the effectiveness of future aquatic habitat rehabilitation projects. In addition to increasing in-channel habitat, watershed-scale improvements of water quality might be necessary to ensure rehabilitation strategies are successful.