Organophosphorus pesticides such as Dimethoate, Malathion, and Tamaron are among the most commonly used insecticides in agriculture in Egypt. The toxic effects of these pesticides on aquatic ecosystem organisms were studied using several freshwater microalgae species, including Chlamydomonas globosa, Chlorella vulgaris, Scenedesmus obliquus, Microcystis aeruginosa, Nostoc punctiforme, Gomphonema olivaceum, Navicula submuralis, and Nitzschia sp., along with the standard toxicity microalga, Raphidocelis subcapitata. The lethal concentration (LC50) and linear regression analysis were used to investigate the relationship between pesticide concentration, protein content, and chlorophyll a (Chl a) in the freshwater microalgae. The relative sensitivity of Chl a and protein content varied depending on the pesticide concentration and the specific microalgal species tested. The total protein content of all tested diatom species was more sensitive to Dimethoate than their Chl a levels. In contrast, the protein content of other algae species, particularly the blue-green algae M. aeruginosa and N. punctiforme, was more tolerant to Dimethoate than Chl a. For Malathion, Chl a in most of the studied microalgae was more resistant than the protein content, whereas for Tamaron, protein content was more resistant than Chl a. Both Chl a and protein content can serve as relevant indicators for assessing pesticide effects on microalgae, but they may provide distinct insights. The toxicity of pesticides to microalgae cannot be fully determined by protein content alone. Future research on pesticides should aim to clarify their specific impacts on algae and explore a more comprehensive understanding of their effects.