Soil washing with chelators is a promising and efficient method of remediating metals-contaminated soils. However, the toxicity of residual metals and the effects on soil microbial properties have remained largely unknown after washing. In this study, we employed four biodegradable chelators for removal of metals from contaminated soils: iminodisuccinic acid (ISA), glutamate-N,N-diacetic acid (GLDA), glucomonocarbonic acid (GCA), and polyaspartic acid (PASP). The maximum removal efficiencies for Cd, Pb, and Zn of 85, 55, and 64% and 45, 53, and 32% were achieved from farmland soil and mine soil using biodegradable chelators, respectively. It was found that the capacity of ISA and GLDA to reduce the labile fraction of Cd, Pb, and Zn was similar to that of the conventional non-biodegradable chelator ethylenediaminetetraacetic acid (EDTA). The leachability, mobility, and bioaccessibility of residual metals after washing decreased notably in comparison to the original soils, thus mitigating the estimated environmental and human health risks. Soil β-glucosidase activity, urease activity, acid phosphatase activity, microbial biomass nitrogen, and microbial biomass phosphorus decreased in the treated soils. However, compared with EDTA treatment, soil enzyme activities distinctly increased by 5-94% and overall microbial biomass slightly improved in the remediated soils, which would facilitate reuse of the washed soils. Based on soil toxicity tests that employed wheat seed germination as the endpoint of assessment, the washed soils exhibited only slight effects especially after ISA and GLDA treatments, following high-efficiency metal removal. Hence, ISA and GLDA appear to possess the greatest potential to rehabilitate polluted soils with limited toxicity remaining.