Bacterial infections caused by methicillin-resistantStaphylococcus aureushave seriously threatened public health. There is an urgent need to propose an existing regimen to overcome multidrug resistance of MRSA. A unique class of novel anti-MRSA thiazolylketenyl quinazolinones (TQs) and their analogs were developed. Some synthesized compounds showed good bacteriostatic potency. Especially TQ4was found to exhibit excellent inhibition against MRSA with a low MIC of 0.5 μg/mL, which was 8-fold more effective than norfloxacin. The combination of TQ4with cefdinir showed stronger antibacterial potency. Further investigation revealed that TQ4,with low hemolytic toxicity and low drug resistance, was not only able to inhibit biofilm formation but also could reduce MRSA metabolic activity and showed good drug-likeness. Mechanistic explorations revealed that TQ4could cause leakage of proteins by disrupting membrane integrity and block DNA replication by intercalated DNA. Furthermore, the synergistic antibacterial effect with cefdinir might be attributed to TQ4with the ability to induce PBP2a allosteric regulation of MRSA and further trigger the opening of the active site to promote the binding of cefdinir to the active site, thus inhibiting the expression of PBP2a, thereby overcoming MRSA resistance and significantly enhancing the anti-MRSA activity of cefdinir. A new strategy provided by these findings was that TQ4, possessing both excellent anti-MRSA activity and allosteric effect of PBP2a, merited further development as a novel class of antibacterial agents to overcome increasingly severe MRSA infections.