Gulf sturgeon Acipenser oxyrinchus desotoi is listed as 'threatened' under the US Endangered Species Act throughout its range in the northern Gulf of Mexico, with Mobile Bay, Alabama, USA, as the recognized break between eastern and western populations. Population recovery requires protection of the species and its critical habitat. We examined Gulf sturgeon physical habitat attributes and infaunal macrobenthic prey density and composition both spatially and seasonally relative to acoustically tagged Gulf sturgeon occurrence in the Pascagoula River estuary. Gulf sturgeon occupancy patterns indicated that adults move quickly through the system during fall and spring compared to longer but more spatially and temporally variable occupancy for juveniles and sub-adults in both seasons; sub-adults exhibited a less spatially and temporally variable occupancy pattern. We found significant differences in physical habitat and macrobenthic density characteristics that partially explained Gulf sturgeon spatial and temporal occupancy patterns. Direct comparisons of physical drivers (% silt, depth, particulate organic carbon) and macrobenthic density patterns (BEST procedures) were significantly correlated (p < 0.01) but weak (global R = 0.277) and suggest alternate hypotheses to better explain the differential estuarine habitat use patterns. The most parsimonious explanation with multiple weights-of-evidence suggests reduced use of the eastern distributary habitat by Gulf sturgeon based on synergistic effects of urbanization and industrialization such as bulkheading, channelization, dredging and related maintenance activities, and beach re-nourishment, all of which occur almost exclusively in the east zone of the estuary.