Water level management in Mississippi River Pool 25 differentially influences off-channel habitats in the mid-pool and lower pool. Hydrologic models indicate lower pool off-channel habitats dry with greater frequency and duration compared to similar habitats at mid-pool. We examined the influence of this contrasting hydrology on substrate characteristics, organic matter, macroinvertebrate, and fish communities in off-channel habitats during 2001--2003. Benthic organic matter standing stocks were stable in mid-pool habitats but lower pool values were variable because of annual differences in moist-soil vegetation production. Generally, small-bodied and multivoltine invertebrate taxa had high community biomass and dominated lower pool habitats, whereas longer-lived and large-bodied taxa were more abundant and had higher community biomass in mid-pool habitats having longer hydroperiods. Fish communities were dominated by cyprinids in both habitats, and mid-pool habitats tended to be higher in overall species richness. Unique fish taxa were collected in each pool, with primarily rheophilic forms in mid-pool habitats and limnophilic forms in lower pool habitats. Results indicate that contrasting hydrology associated with a mid-pool control point directly and indirectly influences biological communities in off-channel habitats. Further, management regimes that promote hydrologic diversity in off-channel habitats may enhance biological diversity at larger spatial and temporal scales.