A new series of pyrazolo[3,4-b]pyridine compounds (3a,b–9a-c) was synthesized starting with 2-oxo-6-phenyl-1,2-dihydropyridine-3-carbonitriles 1a,b which converted to their 2- chloro analogues 2a,b. By further treatment of of 2a,b with hydrazine hydrate, the key intermediates 3-amino-pyrazolopyridine derivatives 3a,b were afforded. Whereas, the target 3-substituted-pyrazolopyridine derivatives (4a-d–9a-c) were obtained through treatment of 3a,b with different reagents. All the new compounds were evaluated as antimicrobial agents against six bacterial and six fungal strains. The most potent antimicrobial activity was showed by compounds (3a, 3b, 4a, 4d, 6a, 6c, 9a and 9c) with MIC values range (2-32) μg/mL. Moreover, the most active compounds were selected to be evaluated for their inhibition activity against the resistant bacteria methicillin-resistant Staphylococcus aureus (MRSA). In addition, the inhibitory activity of the potent compounds against dihydrofolate reductase (DHFR) was evaluated compared with Trimethoprim (TMP) as a reference DHFR inhibitor. The most potent inhibition of the target enzyme was also showed by compounds 4d, 6c and 9c of IC50 values 0.72, 0.95 and 1.09 µM, compared with the IC50 value 5.54 µM of TMP. Also, molecular docking study showed that compounds 4d, 6c and 9c having the most binding affinity in DHFR active site.