Abstract The micellar in situ extraction of hydrophobic compounds from microalgae cultures ( Chlamydomonas reinhardtii , Chlorella vulgaris , Scenedesmus obliquus ) using nonionic surfactants (Triton X-114, Tergitol TMN 6, Tergitol 15-s-7) was studied in lab- and pilot scale. Therefore, phase separation behavior of the cloud point systems as well as the biocompatibility of the surfactants was investigated. It was shown that algae cells are concentrated in the aqueous phase, whereas the hydrophobic compounds (fatty acids) are enriched in the micellar phase. The thermodynamic model COSMO-RS was used to calculate partitioning coefficients of fatty acids between the phases. The biocompatibility of the surfactants applied was studied by monitoring the photosynthetic activity of algae upon contact with surfactants using puls amplitude modulation. Significant differences in growth and the photosynthetic activity of different algae strains after exposure to surfactants were determined. Whereas the photosynthetic activity of C. reinhardtii decreased rapidly upon contact with surfactants, the photosynthetic activity of S. obliquus , was 90% in comparison to a control after exposure for 240 min to all tested surfactants. C. vulgaris showed an intermediate decrease. Thus, besides extraction, surfactants might be applied to control the growth of certain algae enabling the monoalgal culturing in outdoor cultivation of microalgae since the growth of the less surfactant-tolerant microalgae could be suppressed. Overall, the results indicate that a micellar extraction with nonionic surfactants offers the opportunity for an in situ extraction of hydrophobic substances directly from the culture medium and might be a promising alternative to a classic solvent extraction of dried algal biomass.