The acute Microtox toxicity of the water accommodated fraction (WAF) of six commercial soybean biodiesel/petrodiesel blends was investigated at different oil loads. We analyzed five fatty acid methyl esters (FAMEs), C10-C24 n-alkanes, four aromatics, methanol, and total organic carbon (TOC) content. At high oil loads, the WAFs' toxicity was significantly higher for blends containing biodiesel. At the lowest load, the WAFs' toxicity decreased almost linearly with decreasing biodiesel in the blend. At intermediate loads, the WAFs of all the blends appeared to have a similar toxicity. Analysis of WAFs confirmed the presence of autoxidation byproducts of FAMEs at high oil loads. Pure unsaturated FAMEs and n-alkanes were nontoxic when present in water at their reported solubility limits. However, 24-h equilibrated WAFs of pure FAMEs were highly toxic for C18:1 and C18:3, but not for C18:2. The authors concluded that at high oil loads, the acute toxicity of the WAFs was caused by FAMEs' autoxidation byproducts, whereas at low oil loads, the toxicity appeared to be caused primarily by the aromatic compounds present in petrodiesel. The addition of a synthetic antioxidant in biodiesel did not appear to affect the concentration of autoxidation byproducts in the WAF but resulted in a slight decrease in its toxicity. The major autoxidation byproducts identified in the WAF of commercial biodiesel were present neither in the WAFs of pure unsaturated FAMEs nor in the WAF of a different soybean biodiesel that was transesterified in our laboratory, which was nontoxic. We concluded that the process of transesterification of biodiesel might be a more critical factor in determining the aquatic toxicity of the fuel than the source of feedstock itself.