Abstract The synthesis of 4- Hydroxy quinolin-2-one derivatives by three deferent synthetic pathways and the formyl substitution on the 3 rd position one heterocyclic quinoline ring. The Schiff base reaction reacts with 4-Hydroxy-3-formylquinolin-2-one and form twelve different derivatives by using four different hydrazine derivatives. These derivative show satisfactory antimicrobial and anti fungal activity. The newly synthesized compounds were screened for their antibacterial activity against B. subtilis, S. aureus, E.coli and K. pneumonia by cup plate method. A control was also prepared for the plates in the using solvent DMSO. Activity of each compound was compared with ciprofloxacin as standard. The newly synthesized compounds were also screened for their antibacterial activity against A. nige and C. albicans by cup plate method. A control was also prepared for the plates in the using solvent DMSO. Activity of each compound was compared with Griseofulvin as standard. The investigation of antibacterial and antifungal screening data revealed that all the tested compounds and showed moderate to good inhibition at 50-100 µg/ml in DMSO. The compound P1-a, P1-b, P2-a, P2-b, P3-a and P3-c showed comparatively very good activity against all the bacterial strains. The good activity is attributed to the presence of pharmacologically active phenyl hydrazones and semicarbazone on position 3 on quinoline ring. Presence of phenyl group on the position-1 also increases the antimicrobial activity. A new series of Substituted 4-Hydroxy quinoline-2-one derivatives have been synthesized. The research study reports the successful synthesis and antimicrobial activity of new 4-Hydroxy-3-substituted-1-H/Methyl/Phenylquinolin-2-one carrying biologically active groups. Their antimicrobial activity study revealed that all the compounds tested showed moderate to very good antibacterial and antifungal activities against pathogenic strains.