Background: In the present research work a series of novel quinoxaline thiosemicarbazide derivatives were synthesized by substitution of some aromatic aldehydes and their antimicrobial evaluation against various microbial strains with molecular docking studies. Methods: Lead molecule (1E, 4E)-1-(7-chloro-3-isopropyl quinoxaline-2(1H)-ylidene) thiosemicarbazide was synthesized and condensed with various aromatic aldehydes to synthesize derivatives. All derivatives (Va-Vf) were characterized by IR., NMR __ampersandsignamp; Mass spectroscopy. The synthesized derivatives were evaluated in vitro for antibacterial and antifungal activities using the agar dilution method. Molecular docking studies of the derivatives were performed against E.coli DNA gyrase B and topoisomerase IV to find out essential binding sites against target protein PDB: 1AJ6 and 1S14 respectively. Results: Compounds Vb, Vc, Ve__ampersandsignamp;Vf exhibited potent antibacterial and antifungal activity. Compounds, Vb and Vc were found to exhibit more potent activity against Gram __ampersandsignndash;Ve, bacterial strains at MIC 0.19 __ampersandsignmicro;g/ml whereas compound Ve and Vf showed potent activity against Gram +Ve bacterial strains and fungal strains at MIC 0.19 __ampersandsignmicro;g/ml and 0.78 __ampersandsignmicro;g/ml respectively. The docking studies revealed that all the compounds exhibit extensive binding to the active pockets of E.coli DNA gyrase B and topoisomerase IV. The compound Vb and Ve exhibit interactive binding energy -8.0 and -8.3 Kcal/mole to the active pocket site of E.coli DNA gyrase and -8.2 and 7.9 Kcal/mole to the active pocket site of topoisomerase IV respectively. Conclusion: In terms of SAR study, it was revealed that the activity profile against microbial strains was altered with electronic effects like electron-withdrawing or donating substitutions in aromatic aldehydes substituted quinoxaline thiosemicarbazide derivatives.