Municipal solid waste incineration (MSWI) fly ash produced in waste-to-energy plants possesses a serious threat to human health. Although the traditional methods including toxicity characteristic leaching procedure and sequential extraction approach can partially evaluate the reduction of heavy metals leaching from thermally treated MSWI fly ash, the potential threat towards organisms is frequently ignored in previous literature. Considering this, herein we systematically assess the cytotoxicity of heat-treated samples using multiple cells from different biological tissues/organs for the first time. The results indicate that the leachability and transferability of heavy metals are declined after treatment. The biological assays demonstrate that the leachates from the treated residues induce lower phytotoxicity and cytotoxicity compared with the original samples. Moreover, according to the cellular responses of multiple cells to the leachates, normal cells (MC3T3-E1, HUVEC, and L929) are more tolerant to the leachates than cancerous cells (4T1, MG63), and the skin fibroblasts (L929), which often interact with the external circumstance, have the best cellular tolerance. This work provides a novel platform to determine the potential biosecurity of MSWI fly ash-derived products towards organisms, when they are served as secondary building materials in the constructional industry that may be contact with animals and human beings.