Various novel optically active substituted aryl 2-Azetidinone 4a-j have been prepared from corresponding Schiff’s bases 3a-j and chloroacetyl chloride in presence of glacial acetic acid in benzene using Dien and Stark apparatus. Structure of the synthesized compound was confirmed by Spectral data (IR, 1H NMR) and elemental analysis. All the newly synthesized compounds were evaluated for their antimicrobial activities. Investigation of antimicrobial activities of compounds was done by Broth dilution method used for the determination of minimum inhibitory concentration. The synthesize compounds were screened for antibacterial against gram-positive bacteria [Staphylococcus aureus (MTCC96), Streptococcus pyogenes (MTCC442)] and gram-negative bacteria [Escherichia coli (MTCC443), Pseudomonas aeruginosa (MTCC424)] and antifungal against Candida albicans (MTCC227), Aspergillus niger (MTCC282) and Aspergillus clavatus (MTCC1323).The compounds showed good antibacterial activity but less active against fungal strain used.