The biologically active compounds (fatty acids, pigments, phenolics, and flavonoid content) were studied in supercritical fluid extracts from the biomass of marine (Ulva clathrata, Cladophora glomerata, Polysiphonia fucoides, and their multi-species mixture) and freshwater (C. glomerata) macroalgae. Different extraction techniques were used in order to compare differences in the biologically active compound composition of the macroalgal extracts. The results indicated that the saturated and unsaturated fatty acids ranged from C9:0 to C22:0. The analysis of differences in the composition of unsaturated to saturated fatty acids in extracts showed that palmitic acid (C16:0) and oleic acid (C18:1, n-9) reached the highest value not only in marine monospecies and multi-species biomass but also in the freshwater macroalga C. glomerata. When comparing the similarity between the concentration of fatty acids and the ratio of the concentration of unsaturated fatty acids to saturated in macroalgal extracts, we found small but not statistically significant variations in values between years (up to 10%). This is acceptable for applications as a stable raw material for industrial purposes. Significantly higher values of fatty acids, carotenoids, and chlorophylls were obtained in the case of SC-CO2 extraction. The active ingredients of polyphenols, possessing antioxidant activity ranged from approximately 2–4%. Moreover, flavonoids represented less than 10% of the total content of polyphenolic compounds. The extraction efficiency of polyphenols was higher from a mixture of marine algae for the ultrasound-assisted extraction compared to freshwater. All these findings show that marine and freshwater macroalgae, as a raw material, have the optimal biologically active compounds composition for cosmetics.