This study provides a first attempt of dose-response analysis and margin of safety using Escherichia coli DH5alpha, Bacillus subtilis as indicator microorganisms to put forward, in general terms and explanations, the toxicity rankings of various ashes of municipal solid waste incinerator (MSWI) for feasibility in further applications. Since the MSWI ash often contains cations of Si, Ca, Al and Fe, it is frequently considered to be recycled for construction building-materials. Growth inhibition of E. coli DH5alpha occurred at concentrations over 0.156, 0.625 and 0.0195 g/L for bottom ash (BA), cyclone ash (CA), scrubber ash (SA), respectively, suggesting the toxicity ranking of SA>BA>CA. In contrast, except for SA (ca. 0.313 g/L), almost same inhibitory levels of ashes to cell growth were also observed in Bacillus subtilis. Evidently, biotoxicity responses were strongly dependent upon the characteristics of indicator microorganism. Based on DH5alpha, the margins of safety (MOS) were thus 0.195, 1.56 and 6.25 mg/L for SA, BA and CA, respectively. Nearly identical levels of MOS were also suggested by B. subtilis, except for SA (3.13 mg/L). Although MSWI residual ashes qualified EPA's standard test of Toxicity Characteristic Leaching Procedure (TCLP), they might still contain other toxic residues (e.g., chloride ions and/or anions) to cause existing toxicity as indicated in this toxicity study.