Two levels Plackett-Burman experimental design was employed to screen the main factors that significantly affect the synthesis of bioactive silver nanoparticles. Scenedesmus obliquus ethanol extract, Scenedesmus obliquus dried algal powder and Spirulina platensis aqueous extract were the most favorable reducing and capping agents. Also the high pH value (10), rpm (150), high concentration of AgNO3 (4 mM) and dark conditions had the highest significant influence on biosynthesis of active nanoparticles. Characterizations of the formed AgNPs were approved by UV–Visible Spectroscopy, Transmission Electron Microscopy and Fourier Transform Infrared spectroscopy. The nanoparticles were varied in sizes and shape that ranged between 2.83 and 27.17nm, depending on the organism used and the method of biosynthesis. Valuable inhibitory effects were showed by all bio-synthesized nanoparticles against four tested Gram- positive and Gram- negative bacteria, except that formed by Spirulina platensis powder. Moreover, the nanoparticles exhibited wide range of antifungal activities. AgNPs prepared from ethanol extract of Spirulina platensis was the most active against HepG2 and MCF-7 cell lines, with IC50values 62.1 and 56.2 ug/mL, respectively. The obtained results, indicated the advantages of AgNPs from microalgae and possible industrial applications as a source of antifungal, antibacterial, and antitumor formulation.