Background Sanitizing the operating theatres (OT) is important to minimize risk of post-operative infections. Disinfection procedures between one operation and another is less aggressive than final cleaning procedures, at the end of the day. Aim was assessing the difference of contamination: i) between different levels of disinfection; ii) before and after the use of a UVC Device (UVC-D). Methods Between December 2019/February 2020 a cross sectional study was conducted in OT in a real clinical context. 94 Petri dishes (PD) were used in 3 OT. Three different sanitation levels (SL1-3) were compared pre- and post-use of UVC-D: i) No cleaning after surgery (SL1); ii) after in-between cleaning (SL2); iii) after terminal cleaning (SL3). UVC-D was employed for 6 minutes, 3 minutes per bed side. PD were incubated at 36 °C and colony forming unit (CFU) counted at 48h. Descriptive statistic, Wilcoxon and Mann-Whitney tests were performed to assess the contamination levels in total, pre/post use of UVC-D, and between different sanitation levels, respectively. Results In total we had a mean of 3.39 CFU/PD (C.I. 2.05 - 4.74) and a median of 1 CFU/PD (Min. 0 - Max. 39), after UVC-D use we had a mean of 2.20 CFU/PD (C.I. 0.69 - 5.09) and a median of 0 CFU/PD (Min. 0 - Max. 133). The UVC-D led to a significant reduction of CFU (p < 0.001). Without UVC-D we had a significant CFU drop (p < 0.05) between SL1 and SL3. Using UVC-D, we observed significant reductions of contamination (p < 0.05) between SL3 and SL1. Comparing SL1 (median 0) post UVC-D use vs SL2 pre UVC-D use (median 0.5), and SL2 post UVC-D use (median 0) vs SL3 pre UVC-D use (median 1) we had a significant reduction of contamination (p < 0.05). Conclusions UVC-D improved environmental contamination in any of the three sanitation levels. Furthermore, the use of UVC-D alone was better than in-between and terminal cleaning. Although these encouraging results, the cleaning procedures executed by dedicated staff has to be considered. Key messages UVC are efficient to decrease contamination in operating theatres regardless of sanitation levels. The additional use of UVC technology to standard cleaning procedures significantly improves sanitation levels.