The 1, 3, 4-Oxadiazole nucleus offers a wide range of applications in hetero cyclic chemistry, including antimicrobial medicine. A series of the 2, 5-disubstituted 1, 3, 4-Oxadiazole derivatives were designed and study was performed against the ergosterol biosynthesis as an antimicrobial target. The drug-likeness properties of the designed compounds were predicted. All the designed compounds showed good ADME properties and investigated for CYP51 inhibitory activity. According to molecular docking studies, all compounds showed better interaction with target protein and could be the potent inhibitor of ergosterol biosynthesis. The designed 2, 5-disubstituted 1, 3, 4-Oxadiazole derivatives analogs could be safer and more or equivalent effective antimicrobial agents.