The qualitative and quantitative composition of carotenoids in the diatom microalga Phaeodactylum tricornutum cultivated under different temperature and light conditions was studied. It was revealed that the carotenoid composition of microalga is represented by 5 fractions: β-carotene, fucoxanthin, diadinoxanthin, diatoxanthin, and diadinochrome. Fucoxanthin was represented by trans- and cis-isomeric forms. It is shown that carotenoid accumulation depends on conditions during cultivation. Fucoxanthin maximum accumulation occurs at 30 °C and constant illumination 4.5 klx.