Recombinant single-chain fragment variable anti-idiotypic antibodies were produced to represent the internal image of HM-1 killer toxin and were used as novel and effective antifungal agents to inhibit in vitro β-1,3-glucan synthase and cell growth. The mechanism of cytocidal activity of anti-idiotypic antibodies was investigated and was compared with the actions of aculeacin A and papulacandin B, the most common antibiotics acting as β-1,3-glucan synthase inhibitors. The degree of inhibition of β-1,3-glucan synthase by both antibodies and antibiotics were examined for yeasts Saccharomyces cerevisiae A451, Cryptococcus albidus NBRC 0612 and Candida albicans IFM 40215. Although the mechanism of actions of the anti-idiotypic antibodies and antibiotics seems identical, the IC₅₀ values for the various yeasts used in this study confirmed that anti-idiotypic antibodies could be used as more effective fungal β-1,3-glucan synthase inhibitors than those of antibiotics.