In situ click chemistry is a target-guided synthesis technique for discovering highly potent enzyme inhibitors by assembling azides and alkynes into triazoles inside the affinity site of a target enzyme. We here review research aimed at the rapid discovery of a novel and potent inhibitor of bacterial chitinases using in situ click chemistry. Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and anti-asthmatics. Argifin, which has been isolated and characterized as a cyclopentapeptide natural product by our research group, shows strong inhibitory activity against chitinases. Via a combination of efforts to develop a useful chitinase inhibitor from an azide-bearing argifin fragment and application of the chitinase template in situ click chemistry with a library of alkynes, we rapidly obtained a very potent and novel 1,5-disubstituted triazole inhibitor of Serratia marcescens chitinase (SmChi) B. The new inhibitor expressed a 300-fold increase of inhibition against SmChiB compared to that of argifin. We also succeeded in obtaining crystal structures of a chitinase complexed with an azide inhibitor and an O-allyl oxime fragment as a mimic of a click partner, revealing an elegant mechanism for accelerating syn-triazole formation of ‘in situ click chemistry’, thereby allowing generation of its own distinct inhibitor. This represents the first example of expression of a pre-triazole state of ‘in situ click chemistry’. This work exemplifies the benefits of ‘in situ click chemistry’-approach in efforts to produce novel and reliable inhibitors.