The new 1,3,4-oxadiazolylquinoxaline ring system was synthesized by the condensation of the quinoxaline 1,4-dioxide acid hydrazide 6 with acetyl chloride in one-pot synthesis. In which the hydrazide was refluxed in excess of acetyl chloride, acetic anhydride or acetic acid in the presence of phosphorus oxychloride forming the 1,3,4-oxadiazolylquinoxaline ring system. Molecular modeling studies have been performed to evaluate their recognition at the hDHFR binding-pocket as potential hDHFR inhibitors. The antibacterial properties of these compounds showed reasonable activities towards gram negative bacteria, Escherichia coli. Compound 20 performed appropriate complementarity study rather well; the conserved amino acids of hDHFR enzyme imitating the MTX docking pattern. Besides, compound 20 showed highest antibacterial activity against E. coli in comparison with the well known bacteriostatic chloramphenicol; the pyrazolyl analogues also have moderate affinity toward the applied bacteria.