The combined impact of temperature and light spectra on the fatty acid (FA) composition in microalgae has been sparsely investigated. The aim of this study was to investigate the interactions of light and temperature on the FA composition in Acutodesmus obliquus. For this purpose, A. obliquus was cultivated with different temperatures (20, 30, and 35°C), as well as broad light spectra (blue, green, and red light). Growth and FA composition were monitored daily. Microalgal FA were extracted, and a qualitative characterization was done by gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS). Compared to red light, green and blue light caused a higher percentage of the polyunsaturated fatty acids (PUFA) 16:4, 18:3, and 18:4, at all temperatures. The highest total percentage of these PUFA were observed at the lowest cultivation temperature and blue and green light. These data imply that a combination of lower temperatures and blue-green light (450–550 nm) positively influences the activity of specific FA-desaturases in A. obliquus. Additionally, a lower 16:1 trans/cis ratio was observed upon green and blue light treatment and lower cultivation temperatures. Remarkably, green light treatment resulted in a comparably high growth under all tested conditions. Therefore, a higher content of green light, compared to blue light might additionally lead to a higher biomass concentration. Microalgae cultivation with low temperatures and green light might therefore result in a suitable FA composition for the food industry and a comparably high biomass production.