An efficient one-pot synthesis in multi-component system (MRCs) for the preparation of pyrazolo[3,4-b]pyridine derivatives from the reaction of 5-amino-1-phenyl-3-(pyridin-3-yl)-1H-pyrazole with 4-anisaldehyde and p-substituted β-ketonitriles or with pyruvic acid and some aromatic aldehydes in acetic acid medium. The reactions were carried out by two different techniques, conventional heating and microwave irradiation. These compounds were screened for their antibacterial activity against Gram-positive bacteria (Bacillus), Gram-negative bacteria (Escherichia coli, Enterobacter cloaca and serratia) and antifungal activity against Fusarium Oxysporum and Penicillium expansum. Also, among the synthesized compounds 4a-f tested for antitumor activity against liver cell line. Compounds 6-(4-Fluorophenyl)-4-(4-methoxyphenyl)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4e) and 4-(4-Methoxyphenyl)-1-phenyl-3,6-di(pyridin-3-yl)-1H-pyrazolo[3,4-b] pyridine-5-carbonitrile (4a) showed the highest activity.