Soil contaminated with trivalent chromium (Cr3+) induced manganese (Mn) mobilization potentially leading to increased ecological toxicity. Although Cr3+ is relatively immobile in soil, its interaction with soil minerals indirectly increases Mn bioavailability, which poses risks to soil organisms. The objective of this study was to assess the bioavailability and potential ecological toxicity of Mn and Cr in Cr3+ contaminated soil using chemical extractions, earthworm (Eisenia fetida) toxicity tests, and dehydrogenase activity (DHA) as bioindicators. The mitigation of Mn toxicity in Cr contaminated soil was evaluated following amendments with biochar and calcium carbonate (CaCO3). Both amendments increased soil pH and reduced bioavailable Mn concentrations by 44–100% through sorption and pH induced immobilization. While bioavailable Cr concentrations also decreased, the effect was less significant because Cr was originally immobile in the soil. Earthworm survival and loss in biomass was not significantly affected by biochar treatment. Dehydrogenase activity was enhanced in biochar and CaCO3 amended soils, indicating improved microbial activity. Principal component analysis confirmed that biochar and CaCO3 amendments reduced bioavailability and metal toxicity to levels comparable to uncontaminated soils. The assessment of bioavailability through chemical extraction and toxicity using earthworm and DHA showed similar trends in this experiment. However, high amounts of biochar may negatively affect earthworms, and a holistic approach is required to effectively evaluate amendments for mitigating metal toxicity in soils.