AimsThe purpose of this study was the genotypic and phenotypic characterization of 57 strains of Staphylococcus epidermidis isolated from cleanroom environments, based on their biofilm formation and antimicrobial resistance profiles. Methods and ResultsBiofilm formation was investigated using real-time PCR (icaA, aap, bhp genes), the Congo red agar method and the crystal violet assay. The majority of the strains (597%; 34/57) did not form biofilms according to the crystal violet assay, although the biofilm-associated genes were present in 947% (54/57) of the strains. Of the biofilm formers (404%; 23/57), 391% (9/23) have been identified as strong biofilm formers (>4x crystal violet absorbance cut-off). Resistance to a commercial disinfectant and its quaternary ammonium active component, didecyl-dimethyl-ammonium chloride (DDAC), was determined according to minimum inhibitory concentrations (MICs) and the presence of the qac (quaternary ammonium compound) genes. More than 95% (55/57) of the Staph. epidermidis strains had the qacA/B and qacC genes, but not the other qac genes. The MICs for the disinfectant and DDAC varied among the Staph. epidermidis strains, although none were resistant. ConclusionsAlthough 596% of the Staph. epidermidis strains did not form biofilms and none were resistant to DDAC, more than 94% had the genetic basis for development of resistance to quaternary ammonium compounds, and among them at least 140% (8/57) might represent a high risk to cleanroom hygiene as strong biofim formers with qacA/B and qacC genes. Significance and Impact of the StudyTo assure controlled cleanroom environments, bacterial strains isolated from cleanroom environments need to be characterized regularly using several investigative methods.