BACKGROUND: The formation of biofilms, which is an important step in bacterial colonization, can be inhibited by deoxyribonuclease (DNase)-mediated breakdown of extracellular DNA. We have recently demonstrated that epidermal keratinocytes strongly express DNase1-like 2 (DNase1L2) in a differentiation-associated manner. OBJECTIVES: To determine whether enzymatically active DNase1L2 is present in human stratum corneum and whether it is able to suppress bacterial biofilm formation. METHODS: DNase1L2 was extracted from normal human stratum corneum, immunocaptured and incubated with plasmid DNA. DNA hydrolysis was monitored by gel electrophoresis and ethidium bromide staining. The effect of DNase1L2 on biofilm formation was assayed by cultivation of Pseudomonas aeruginosa and Staphylococcus aureus in the presence or absence of purified recombinant DNase1L2 in microtitre plates and subsequent quantification of biofilm-forming bacteria by crystal violet staining. RESULTS: DNase1L2 was found to be present in an enzymatically active form in the stratum corneum of human skin. In an in vitro assay, purified recombinant DNase1L2 efficiently suppressed the formation of biofilms by P. aeruginosa and S. aureus. CONCLUSIONS: Our data suggest that DNase1L2 is a novel component of the innate antimicrobial defence of the epidermis.