The O2 content of the global ocean has been declining progressively over the past decades, mainly because of human activities and global warming. Nevertheless, how long‐term deoxygenation affects macrobenthic communities, sediment biogeochemistry and their mutual feedback remains poorly understood. Here, we evaluate the response of the benthic assemblages and biogeochemical functioning to decreasing O2 concentrations along the persistent bottom‐water dissolved O2 gradient of the Estuary and Gulf of St. Lawrence (QC, Canada). We report several of non‐linear biodiversity and functional responses to decreasing O2 concentrations, and identify an O2 threshold that occurs at approximately at 63 μM. Below this threshold, macrobenthic community assemblages change, and bioturbation rates drastically decrease to near zero. Consequently, the sequence of electron acceptors used to metabolize the sedimentary organic matter is squeezed towards the sediment surface while reduced compounds accumulate closer (as much as 0.5–2.5 cm depending on the compound) to the sediment–water interface. Our results illustrate the capacity of bioturbating species to compensate for the biogeochemical consequences of hypoxia and can help to predict future changes in benthic ecosystems.