Lipid oxidation is a recognized end point for the study of oxidative stress and is an important parameter to describe the mode of micropollutant action on aquatic microorganisms. Therefore, the development of quick and reliable methodologies probing the oxidative stress and damage in living cells is highly sought. In the present proof-of-concept work, we examined the potential of the fluorescent dye C11-BODIPY591/581 to probe lipid oxidation in the green microalga Chlamydomonas reinhardtii. C11-BODIPY591/581 staining was combined with flow cytometry measurements to obtain multiparameter information on cellular features and oxidative stress damage within single cells. First, staining conditions were optimized by exploring the capability of the dye to stain algal cells under increasing cell and dye concentrations and different staining procedures. Then lipid oxidation in algae induced by short- and long-term exposures to the three metallic micropollutants, copper, mercury, and nanoparticulate copper oxide, and the two organic contaminants, diethyldithiocarbamate (DDC) and diuron was determined. In this work we pointed out C11-BODIPY591/581 applicability in a wide range of exposure conditions, including studies of oxidation as a function of time and that it is suitable for in vivo measurements of lipid oxidation due to its high permeation and stability in cells and its low interference with algal autofluorescence.