Perchlorate (ClO4-) is a strong oxidizer, capable of accelerating heavy metal release into regolith/soil. Here, we assessed interactions between ClO4- and serpentine soil to simulate and understand the fate of Ni and Mn and their immobilization with the presence of biochar (BC). A soil incubation study (6 months) was performed using serpentine soil in combination with different ClO4- concentrations (0.25, 0.5, 0.75, and 1 wt.%) and three different amendment rates (1, 2.5, and 5 wt.%) of Gliricidia sepium BC. Bioavailable fraction of Ni and Mn was analyzed using CaCl2 extraction method. An increase of ClO4- concentrations enhanced bioavailability fraction of Ni and Mn. However, BC amendments reduced the bioavailability of Ni and Mn. In comparison, 5% BC amendment significantly immobilized the bioavailability of Ni (68–92%) and Mn (76–93%) compared to other BC amendment rates. Electrostatic attractions and surface diffusion could be postulated for Ni and Mn immobilization by BC. In addition, ClO4- may have adsorbed to BC via hydrogen bonding which may reduce the influence of ClO4- on Ni and Mn mobility. Overall, it is obvious that BC could be utilized as an effective amendment to immobilize Ni and Mn in heavy metal and ClO4- contaminated soil.