Abstract Arbuscular mycorrhizal fungi (AMF) can improve plant tolerance to heavy metal stress in the terrestrial system. However, the detoxification ability of AMF to heavy metals in aquatic systems remains poorly understood. This study investigated the effects of AMF on chromium (Cr) distribution and bioavailability in semi-aquatic habitats under different water depths. Results showed that AMF increased the Cr accumulation in the roots of Iris pseudacorus by 10.0–20.0%. Conversely, Cr concentrations in the shoots of AMF inoculated I. pseudacorus were 21.7–68.4% lower than those in the non-inoculated controls. Besides, Cr concentrations and mass contents in water were decreased by 34.1–35.3% and 15.4–23.3% under the low Cr stress (5 mg kg−1) with AMF inoculation compare to the non-inoculated treatments, respectively. Moreover, Cr (VI) concentrations in substrates under the Cr contents of 5 mg kg−1 were 27.0–44.0% lower than those under the non-inoculated controls. Meanwhile, AMF reduced the Cr bioavailability in substrates under the fluctuating water depth (water depth of 6–3 cm), with an acid-soluble state of Cr decreased by 4.1–5.6%. Furthermore, AMF also enhanced the biomass, nutrient contents (TC, TN, Ca, Mg, Fe, and Mn) in the I. pseudacorus under the fluctuating water depth. Therefore, it provides a possibility that AMF can be used to remove heavy metals from polluted wastewater by semi-aquatic habitats under fluctuating water depth (e.g. tidal flow constructed wetlands).