Five microalgae (Nitzschia sp. S5, Nanofrustulum shiloi D1, Tetraselsmis sp. C6, Tetraselmis sp. Z3, Picochlorum sp. D3) and one cyanobacterium (Euhalothece sp. C1) from the Adriatic Sea as well as three microalgae from the Gacka River (Chlorella vulgaris S2, Clorococcum sp. S1 and Tetradesmus obliquus S3) were isolated and identified. Microalgae were characterized regarding growth kinetics, macromolecular composition, and antimicrobial and antioxidant activity. Microalga Picochlorum sp. D3 showed the highest growth rate, while the highest biomass concentrations were achieved with microalgae Tetraselmis sp. Z3 (0.48± 0.11 g Lˉ¹) and Tetraselmis sp. C6 (0.45± 0.04 g Lˉ¹). Proteins were the most abundant macromolecule in all characterized microalgae (32.83–57.94%, g gˉ¹). Marine microalgae lipids were rich in polyunsaturated fatty acids (eicosapentaenoic, docosahexaenoic, alpha-linolenic), while carotenoids were the most abundant pigments. Methanolic extracts of all investigated microalgae showed good antioxidant activity and strongly inhibited the growth of Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. The nutrient concentration in the growth medium and the growth conditions were optimized for selected microalgae. In fed-batch cultivation of Nitzschia sp. S5 in the optimized growth medium with a nutrient molar ratio of [Si]: [N-NO3]:[PO43-] of 6.67:23.33:1 [mM:mM:mM] biomass concentration was six times higher compared to batch cultivation using an original f/2 medium. The highest biomass concentration of Picochlorum sp D3 was achieved in fed-batch cultivation in a bubble column (1.68 g Lˉ¹). Supplementing the culture with sodium bicarbonate stimulated lipid accumulation resulting in doubled lipid content. Effective self-flocculation (>98 %) of Picochlorum sp. D3 was achieved at pH>11, and flocculation (>97%) using 1100 mg L-1 ferric chloride at pH=9 and 25 mg Lˉ¹chitosan at pH=8. Bioflocculation with the fungi Mortierella isabelina was successfully performed under optimized conditions (microalgae: fungus=1:50 g gˉ¹, pH=2 and T=30°C) with an efficiency of 88.47%. The highest adsorption efficiency of microalgae C. vulgaris S2 cells was achieved with Aspergillus niger. Suitable carbon sources for cultivating freshwater microalgae under mixotrophic and heterotrophic conditions weres glucose, galactose, molasses, and acetic acid. Cultivation of the microalgae C. vulgaris S2 with higher molar ratios of carbon and nitrogen (C:N=10, 30, 50, 75 mol molˉ¹) promoted the lipid accumulation during batch and fed-batch cultivation. Heterotrophic cultivation conditions were more suitable for the biomass growth of freshwater microalgae than mixotrophic conditions. The two-stage batch cultivation of C. vulgaris S2 resulted in a high biomass concentration of 19.31 g Lˉ¹ and productivity of 0.075 g Lˉ¹.