Aim: The aim of the present study is to synthesis and evaluation of anti-microbial activity of novel series of quinoxaline 2, 3 Dione derivatives containing a 2-azetidinone (A1-A5) nucleus. Method: The synthesis of azetidinones by reacting with chloroacetyl chloride in the presence of triethylamine. All the title compounds were tested for their anti-microbial activity against gram-positive (staphylococcus aureus, staphylococcus epidermidis, Bacillus cereus and pseudomonas aereginosa) and gram-negative (Escherichia coli and klebsiella pneumonia) bacteria, fungal activity against (Aspergillus Niger and Aspergillus fumigates) at a concentration of 100,200µg/ml. Result: Antibacterial activity of synthesized compounds was tested against both gram positive and gram negative bacteria and the standard drug used for the study was ciprofloxacin. The chloro, fluro and bromo substitution in three derivatives showed significant antifungal activity when compared to standard. The hydroxy, nitro groups showed mild activity when compared to standard. Conclution: A novel series of quinoxaline 2, 3- Dione derivatives has anti-microbial activity so supplementary exploration has been needed to establish other pharmacological performance of these derivatives and in future it would be a most promising and leading derivatives for several ailments. Keywords: Quinoxaline 2; 3-Dione; Antimicrobial activity; Anti-fungal activity; Schiff bases reactions.