Background: Hospital-acquired infections (HAIs) remain a common problem, which suggests that standard decontamination procedures are insufficient. Thus, new methods of decontamination are needed in hospitals. Methods: We assessed the effectiveness of a no-touch automated disinfection (NTD) system in the decontamination of 50 surfaces in 10 hospital rooms. Contamination of surfaces was assessed with a microbiological assay and an ATP bioluminescence assay. Unacceptable contamination was defined as > 100 colony forming units/100 cm2 in the microbiological assay, and as ≥ 250 relative light units in the ATP assay. Results: When measured with the microbiological assay, 11 of 50 surfaces had unacceptable contamination before NTD, and none of the surfaces had unacceptable contamination after NTD (p < 0.001). On the ATP bioluminescence assay, NTD decreased the number of surfaces with unacceptable contamination from 28 to 13, but this effect was non-significant (p = 0.176). On the microbiological assay taken before NTD, the greatest contamination exceeded the acceptable level by more than 11-fold (lamp holder, 1150 CFU/100 cm2). On the ATP bioluminescence assay taken before NTD, the greatest contamination exceeded the acceptable level by more than 43-fold (Ambu bag, 10,874 RLU). Conclusion: NTD effectively reduced microbiological contamination in all hospital rooms. However, when measured with the ATP bioluminescence assay, the reduction of contamination was not significant.