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Contamination of surfaces by spores of Clostridium difficile is a major factor influencing the spread of healthcare-associated C. difficile infection. The aim of this study was to test the effect of an automated room disinfection system that provides an aerosol of 7.5 % hydrogen peroxide (H2O2) disinfectant, on spores of two different strains of C. difficile, and to evaluate the impact of biological soiling on the efficacy of H2O2 disinfection. The strains used were a C. difficile PCR ribotype 027 and a C. difficile ATCC 9689. Spore suspensions of each strain were applied to ceramic tiles and exposed to aerosolized H2O2 at different locations in a test room. Biological soiling was simulated by bovine serum albumin and sheep erythrocytes. At set time points spores were recovered, plated onto Columbia 5 % sheep blood agar, and surviving bacteria were counted as colony-forming units (cfu). No viable spores of either strain were recovered after a 3 h exposure to gaseous H2O2. Spores located inside a drawer showed recovery of approximately 1E5 cfu/ml for C. difficile ribotype 027 after 1 h. In the presence of organic matter, a more than fivefold log reduction compared with not exposed controls could be observed for spores of either strain tested. Appropriate decontamination of surfaces exposed to spores of C. difficile is challenging for conventional cleaning methods. Aerosolized H2O2 delivered by automated room disinfection systems could possibly improve surface decontamination and thereby reduce transmission of healthcare-associated C. difficile infection. Also in the presence of organic matter H2O2 disinfection appears to be an effective adjunct for decontamination of environmental surfaces.