Abstract: Monochloroacetic acid (MCAA), a disinfection byproduct, is widely present in aquatic environments; however, its toxic mechanisms in aquatic primary producers remain unclear. In this study, using Chlorella pyrenoidosa, Scenedesmus obliquus, Navicula pelliculosa, and Nitzschia palea as test organisms, Fourier transform infrared (FTIR) spectroscopy combined with principal component analysis-linear discriminant analysis (PCA-LDA) was employed to systematically elucidate the molecular-level toxic responses induced by MCAA. The results demonstrated that MCAA induced multi-target damage in algae by triggering membrane lipid peroxidation (evidenced by decreased CH₂/lipid ratio), alterations in protein secondary structure (increased β-sheet/α-helix ratio), and conformational changes in DNA. Notably, distinct response patterns were observed among the different algal species: Chlorella pyrenoidosa displayed a exhibited adaptive responses primarily through enhanced cell wall polysaccharides and membrane lipid remodeling, whereas Scenedesmus obliquus displayed pronounced protein denaturation, membrane damage, and nucleic acid disruption. The diatoms (Navicula pelliculosa and Nitzschia palea) exhibited intermediate responses; however, their higher unsaturated fatty acid content rendered them more susceptible to lipid peroxidation. This study elucidated the toxicity response characteristics of MCAA to algae at the molecular level and speculated on its potential toxic mechanisms of action.