Abstract Capable of oxidizing a wide range of reducing substances, laccase has a great potential for various biocatalytic applications. Extensive research effort is being made for improving laccase's activity, specificity, and stability. We observed that pre-incubating with penicillin led to a significant activation of a Rhizoctonia solani laccase, although penicillin was not a redox substrate of the enzyme. The activation impacted mainly the k cat of the laccase, and the effect was optimal at pH 5. Ampicillin and penicilloic acid were also active on the laccase (although their effect were weaker than that of penicillin), but 6-aminopenicillanic acid, phenylacetic acid, and cephalexin were not active. The apparent effect from penicillin was attributed to a non-redox interaction with the polypeptide backbone of the laccase. The effect was significantly weaker on Myceliophthora thermophila and Trametes villosa laccases.