β-Lactam antibiotics marked the beginning of an era of effective and safe treatment for bacterial infections and remain the most widely prescribed antibacterial agents today. However, the emergence of antibiotic-resistant bacteria threatens a return to the pre-antibiotic era. In particular, bacterial expression of β-lactamases inactivating β-lactam antibiotics presents a challenge in antimicrobial therapy. While inhibitors against β-lactamases have been developed to protect the therapeutic efficacy of β-lactam antibiotics, the clinical use of β-lactamase inhibitors is constrained due to their limited inhibition spectrum and the emergence of inhibitor-resistant β-lactamase variants. As an effort to tackle this issue, here we reviewed the structural and mechanistic features of β-lactamases and their FDA-approved inhibitors. Moreover, mutations in clinically isolated β-lactamases that confer resistance against their inhibitors are compiled. The comprehensive overview offered in this review aims to support and stimulate the design of next-generation β-lactamase inhibitors for combating β-lactamase-mediated antibiotic resistance.