Methicillin-resistant Staphylococcus aureus (MRSA) is a major global challenge due to its resistance to β-lactam antibiotics. This resistance stems from the mecA gene, which encodes penicillin-binding protein 2a (PBP2a). PBP2a enables bacterial survival by catalyzing peptidoglycan synthesis while exhibiting low affinity for β-lactams. Its active site is shielded by protective loops, adopting a closed conformation that limits drug access. These structural changes are regulated by an allosteric site distant from the active site. Given the urgent need for new therapeutic strategies, this review focuses on PBP2a’s conformational dynamics and allosteric regulation in β-lactam resistance. Additionally, it explores ligand interactions that influence PBP2a inhibition and their potential in restoring antibiotic efficacy. Understanding these mechanisms will aid the design of adjuvants targeting the allosteric site, offering a promising strategy to enhance β-lactam effectiveness against MRSA.