The SARS-CoV-2 main protease (Mpro) plays a pivotal role in viral replication and is a validated therapeutic target. Previous work has revealed that derivatives of clinically used penicillin antibiotics can inhibit Mproin vitro through non-covalent binding at the active site or formation of stable acyl-enzyme complexes via reaction of the nucleophilic Cys145 with the penicillin β-lactam ring. Here we demonstrate that cephalosporin derivatives are substantially more potent inhibitors of isolated Mpro than the reported penicillin derivatives. Structure-activity relationship and computational studies reveal the importance of appropriate cephalosporin C3 substitution in modulating inhibition potency and mechanism, with both covalent reaction with Cys145 and non-covalent active site binding being observed. Cephalosporin derivatives also show potential for inhibiting SARS-CoV-2 replication in infected cells. The combined results imply that the cephalosporin scaffold may be of use for development of antiviral drugs targeting the SARS-CoV-2 proteases and, by implication, other nucleophilic cysteine proteases. They further highlight the potential of bicyclic β-lactams for therapeutic applications beyond their established use as antibiotics inhibiting bacterial nucleophilic serine transpeptidases.