The objective of this study was to analyze the results of microbiological air sampling of operating rooms (OR) over the last two decades at the Sapienza University Hospital of Rome, in order to describe the time trends of contamination levels and to assess any significant changes. Microbiological air sampling carried out in 14 surgical units between 1992 and 2010 were examined. The sampling results have been aggregated into four time periods (prior to 1996, 1996-2000, 2001-2005, 2006-2010) and the time trend of sampling results was analyzed in comparison with the standard reported by ISPESL for OR at-rest (< or = 35 CFU/mc). The same analysis was repeated after stratification by risk level of the OR (high risk (AR) and low risk (BR)). To verify the significance level of the temporal variations in the distribution of results x2 test for trend was performed. There was a significant downward trend in the number of OR with contamination levels higher of the standard (x2 for trend = 8.94, P < 0.025). This reduction mainly regards AR-OR (x2 for trend = 7.33, P < 0.05). The results suggest that the preventive measures performed in AR-OR have been effective. More attention must be given to BR-OR.