Abstract Background Although chlorhexidine allergy has been shown to be mediated by immunoglobulin (Ig) E, few reports investigated the mechanism of chlorhexidine-induced anaphylaxis using basophil activation tests (BATs). Case presentation A 79-year-old man underwent cholecystectomy under general anesthesia. Anaphylaxis was diagnosed based on the clinical symptoms and high serum tryptase and histamine levels. Skin tests showed positive results only for chlorhexidine. Subsequently, BATs demonstrated that the causative agent was likely chlorhexidine. The inhibitory effect of wortmannin, an inhibitor of phosphoinositide 3-kinase, on basophil activation suggested an IgE-dependent mechanism underlying chlorhexidine-induced anaphylaxis. An 89-year-old man underwent inguinal hernioplasty under general anesthesia. Anaphylaxis was diagnosed based on the clinical symptoms and high serum tryptase and histamine levels. Skin tests and BATs with wortmannin were performed, showing similar results to case 1. Conclusions BATs suggested an IgE-dependent mechanism for chlorhexidine-induced anaphylaxis and might be useful for investigating the mechanisms underlying drug-induced anaphylaxis.