We recently developed a chlorophyll a fluorescence method (activated F0 rise) for estimating if a light wavelength preferably excites PSI or PSII in plants. Here, the method was tested in green microalgae: Scenedesmus quadricauda, Scenedesmus ecornis, Scenedesmus fuscus, Chlamydomonas reinhardtii, Chlorella sorokiniana, and Ettlia oleoabundans. The Scenedesmus species displayed a plant-like action spectra of F0 rise, suggesting that PSII/PSI absorption ratio is conserved from higher plants to green algae. F0 rise was weak in a strain of C. reinhardtii, C. sorokiniana, and E. oleoabundans. Interestingly, another C. reinhardtii strain exhibited a strong F0 rise. The result indicates that the same illumination can lead to different redox states of the plastoquinone pool in different algae. Flavodiiron activity enhanced the F0 rise, presumably by oxidizing the plastoquinone pool during pre-illumination. The activity of plastid terminal oxidase, in turn, diminished the F0 rise, but to a small degree.