This study synthesized a series of novel 2-( p -substitutedphenyl)-5-[2-(4-ethylpiperazin-1-yl)acetamido]benzoxazole series, clarified their structures using spectral techniques, and compared the novel benzoxazoles' MICs with those of several reference drugs using standard bacterial and fungal strains as well as drug-resistant isolates. Compounds 3b and 3d exhibited activity closest to that of ampicillin and vancomycin, with an MIC value of 32 μg/mL against the E. faecalis isolate. Furthermore, 3d showed antimicrobial activity very close to ampicillin, with 16 μg/mL inhibiting E. coli and an E. coli isolate. The interactions of the compounds with the DNA gyrase enzyme (PDB ID: 4KTN ) were evaluated using molecular docking, and images of protein-ligand interactions are also shown. The docking study disclosed that the compounds could bind to the DNA gyrase structure. According to the molecular dynamics simulation results, the complexes obtained from the docking were also found to be stable. DFT was used to model the molecular structure of the compounds at the B3LYP/6-311G (d,p) level of theory. In addition to the optimized geometric structures, HOMO and LUMO orbital energies and other chemical parameters obtained from these energies were estimated. MEP studies also examined the compounds' electrophilic and nucleophilic states. According to the HOMO–LUMO analysis, compound 3d has the lowest LUMO value (−1.6876 eV) and exhibits the best antimicrobial activity among the in vitro antimicrobial activity results. Additionally, Mulliken population analyses, NBO analyses, and estimated ADMET profiles of the compounds were calculated.