The mass production and applications of tripropyl phosphate (TPrP) and tributyl phosphate (TBP) have facilitated their widespread distribution in aquatic environments, thereby posing a threat to the ecosystem. Here, the acute toxicity of TPrP and TBP to Microcystis aeruginosa and the underlying mechanisms were investigated. The results demonstrate that both TPrP and TBP can significantly inhibit the growth and reduce cell viability of M. aeruginosa with increasing concentrations and exposure time. Moreover, the treatment with TPrP and TBP result in a notable reduction in the content of chlorophyll a. The content of dissolved organic carbon (DOC) is down-regulated at lower concentrations, and shows a gradual increase with increasing concentrations of TPrP or TBP. Meanwhile, minor discrepancies have been observed in the proportions of DOC components through excitation-emission-matrix (EEM) spectra. The exposure of TPrP and TBP results in the production of excessive reactive oxygen species (ROS) and the increase of antioxidant enzymatic activities, including superoxide dismutase (SOD) and catalase (CAT). TPrP, but not TBP, has been demonstrated to enhance the MDA level, indicating a significant effect on membrane lipid peroxidation. The differences in the respective toxicity mechanisms and biological effects can be attributed to the alkyl chain lengths and physicochemical properties inherent to each compound. Consequently, the study not only offers insights into the acute effects of the two alkyl organophosphate esters on M. aeruginosa, but also provides a scientific basis and framework for assessing their ecological risk in aquatic environments.