A class of new potential antibacterial agents with distinctive pyrimidinone benzimidazole skeleton was developed through nucleophilic substitution and Biginelli reaction starting from urea, ethyl 4‐chloroacetoacetate and various aldehydes. Some target molecules exhibited strong antibacterial activities, especially pyrimidinone benzimidazole hybrid 9e possessed the strongest inhibitory effects on the growth of E. faecalis and P. aeruginosa with a lower MIC value of 1 μg/mL than norfloxacin. Moreover, compound 9e displayed strong antibiofilm capacity, low drug resistance and excellent biosafety toward human red blood cells. Further research revealed that compound 9e could disrupt membrane integrity and cause leakage of cellular components such as proteins and nucleic acids. Meanwhile, compound 9e could decrease lactate dehydrogenase activity, block cell metabolism and interact with DNA in an intercalation manner. ADMET analysis predicated that molecule 9e possessed promising pharmacokinetic properties and good bioavailability profile.