Halogen-substituted para-benzoquinones (p-BQs) are emerging disinfection byproducts known to induce oxidative damage both in vitro and in vivo. However, as ubiquitous oxidation byproducts, the in vivo toxicity and transport mechanism of non-halogenated p-BQs with similar structure of α, β-unsaturated ketones to halogenated p-BQs have not been thoroughly investigated. In this study, the effect of substituents on toxicity and transportation of 2-chloro-1,4-benzoquinone (CBQ) and 2-methyl-1,4-benzoquinone (MBQ) was systematically investigated. The results show that MBQ exhibits slightly lower acute toxicity to zebrafish embryos compared to CBQ. Exposure to both CBQ and MBQ at concentration of 10 μg/L and 100 μg/L significantly increased the levels of reactive oxygen species, and enhanced the activities of total superoxide dismutase, catalase, and glutathione peroxidase, while malformations were primarily observed in the 100 μg/L exposure groups. The varying developmental toxicity was associated with significant upregulation of 10 genes by CBQ compared to only 6 by MBQ. Using the high-resolution mass spectrometry and electron paramagnetic resonance spectroscopy, the hydroxylation of both CBQ and MBQ, and the production of semiquinone radicals and hydroxyl radicals in aqueous environments have been revealed. This study has demonstrated that the toxicity of non-halogenated p-BQs should not be overlooked and contributes to the understanding of the generated radicals, leading to excessive oxidative-stress in vivo.