A class of structurally unique para-aminobenzenesulfonyl oxadiazoles as new potential antimicrobial agents was designed and synthesized from acetanilide. Some target para-aminobenzenesulfonyl oxadiazoles showed antibacterial potency. Noticeably, hexyl derivative 8b (MIC = 1 μg/mL) was more active than norfloxacin against drug resistant MRSA. Compound 8b was able to disturb the membrane effectively and intercalate into deoxyribonucleic acid (DNA) to form a steady 8b-DNA complex, which might be responsible for bacterial metabolic inactivation. Molecular docking indicated that 8b could interact with DNA topoisomerase IV through noncovalent interactions to form a supramolecular complex and hinder the function of this enzyme. These results indicated that hexyl derivative 8b deserved further investigation as a new lead compound.