This paper intend to investigate the effects of biodiesel fuel blends comprising of waste-cooking oil and butanol-diesel (B10W10–B10W40) under steady-state conditions. Both particulate organic carbon (OC) and PM including PM2.5 and PM10 significantly decreased with the increasing percentage of biodiesel fuel blends. The fuel blend of B10W40 also demonstrated the most effective function in reducing the emissions of PM10 and PM2.5 in the volume by 59.4% and 57.7%, respectively. Moreover, the emissions of nitrogen oxides decreased with the blending of B10W10-B10W40 (13.9–28.5%), while the brake specific fuel consumption was substantially increased (5.69–13.4%). The overall biological toxicity of PM10 generated from the fuel tested in this study was determined according to Single Cell Gel Electrophoresis assay in human alveolar basal epithelial A549 cells and micronucleus assay in CHO-K1 cells. In addition, the volume of more than 20% waste-cooking oil (B10W20 and B10W40) significantly reduced diesel-induced genotoxicity in lung cells and micronucleus formation in CHO-K1 cells. Collectively, these results indicated that biodiesel fuel blends with the butanol could be a potential alternative fuels for diesel engines because of its substantial property with a significant reduction of the PM-related genotoxicity and the emissions of PM, particulate OC, and NOX.