Epolactaene is a microbial metabolite isolated from Penicillium sp., from which we synthesized its derivative ETB (epolactaene tertiary butyl ester). In the present paper, we report on the identification of the binding proteins of epolactaene/ETB, and the results of our investigation into its inhibitory mechanism. Using biotin-labelled derivatives of epolactaene/ETB, human Hsp (heat-shock protein) 60 was identified as a binding protein of epolactaene/ETB in vitro as well as in situ. In addition, we found that Hsp60 pre-incubated with epolactaene/ETB lost its chaperone activity. The in vitro binding study showed that biotin-conjugated epolactaene/ETB covalently binds to Hsp60. In order to investigate the binding site, binding experiments with alanine mutants of Hsp60 cysteine residues were conducted. As a result, it was suggested that Cys⁴⁴² is responsible for the covalent binding with biotin-conjugated epolactaene/ETB. Furthermore, the replacement of Hsp60 Cys⁴⁴² with an alanine residue renders the chaperone activity resistant to ETB inhibition, while the alanine replacement of other cysteine residues do not. These results indicate that this cysteine residue is alkylated by ETB, leading to Hsp60 inactivation.