In a trial to discover potential antimicrobial agents against the increasing microbial resistance, eight new Schiff base derivatives of glycine amino acid obtained from the condensation reaction with benzaldehyde were designed and synthesized by conventional approach. The structures of all the synthesized compounds were elucidated using IR, 1H NMR and 13C NMR along with MS spectra data. In addition, the antimicrobial activities of the new compounds were screened with gentamycin and ketoconazole as the control against four significant representative strains including Staphylococcus aureus, Escherichia coli, Aspergillus niger, and Candida albicans, using broth microdilution method. The antimicrobial activity results revealed that most of the tested compounds exhibited significant antibacterial activity. In particularly, compound 5 was found to be the most potent antibacterial agent with MIC value of 18 exhibiting better activity against Staphylococcus aureus than Escherichia coli. Additionally, compounds 4, 6, and 10 displayed potent antibacterial activity with MIC value of 15, 16, and 16 respectively. However, compounds 10 and 11 showed good activity against both bacterial strains along with Aspergillus niger, indicating their broad spectrum of activity. The structure-activity relationship analysis suggested that the electronic and lipophilic factors of aromatic substituents on the amide nitrogen significantly contributed to antibacterial activity. Moreover, the presence of aromatic heterocyclic or heterobicyclic containing more nitrogen atoms considerably influenced the antifungal activity.