Alkaloids are one of the prominent members in the development of new antimicrobial agents. This work discovered a class of alkaloid quinazolone-derived imidazolenones as novel structural type of antibacterial agents with large potential to treat severe bacterial infections in the agricultural and food field. Preliminary bioactive assay displayed that some of the prepared compounds exhibited good inhibition against the tested strains, and cyclohexylimidazole-derived 7-fluoroquinazolone 22a (MIC = 0.002 mmol/L) exhibited a 12.5-fold stronger inhibition than norfloxacin against Escherichia coli ATCC 25922. Further studies revealed that compound 22a not only possessed the ability of rapid bactericidal property and low propensity to develop resistance but also showed low cytotoxic effects toward red blood cells. The preliminary mechanism exploration indicated that compound 22a could cause membrane damage by disrupting bacterial membrane as well as depolarizing the cell membrane. Moreover, compound 22a could insert into DNA, which might hinder the replication of DNA. Molecular docking suggested that compound 22a could bind to gyrase and topoisomerase, which might be due to the suppressed expression of related genes. Meanwhile, compound 22a could disorder the metabolism and stimulate the production of reactive oxygen species to affect bacterial growth. The series of investigations suggested the promise of alkaloid quinazolone-derived imidazolenones as novel multitargeting antibacterial candidates for treatment of bacterial infections. © 2023 SIOC, CAS, Shanghai, & WILEY-VCH GmbH.