Acacia albida, Acacia luederitzii, and Acacia tortilis are dominant acacia species in Botswana and have the potential to rehabilitate the heavy metal degraded environment. To establish this claim, experiments to assess the influence of mycorrhizal inoculation and fly ash amendments on the survival, growth and heavy metal accumulation of these species in mine tailings were conducted. A two-factor (AM inoculation × fly ash) in CRD was done on each of the three Acacia species consisting of four treatments: control (no mycorrhizal, no fly ash coded as − AM/− FA), with mycorrhizal but no fly ash (+ AM/− FA), no mycorrhizal but with fly ash (− AM/+ FA), and with mycorrhizal and with fly ash (+ AM/+ FA). After 24 weeks, results showed that the survival and dry matter yield of all Acacia species were enhanced by 10% with fly ash amendments. However, mycorrhiza inoculation alone improved the survival of A. albida and A. luederitzii but reduced that of the A. tortilis in mine tailings. Fly ash amendments increased the pH of the mine tailings, reduced the availability of Cu, Ni, Pb, Mn and Zn and consequently reduced the concentration of these metals in shoots. On the other hand, it increased the availability of As in the mine tailings. In addition, mycorrhizal inoculation reduced the concentration of these metals in shoots regardless of fly ash amendments. Overall, combined mycorrhizal inoculation and fly ash amendment enhanced the establishment of A. luederitzii in heavy metal-contaminated soils by reducing the heavy metal availability and metal uptake, thus increasing the survival and dry matter yield of plants.