Objective: Healthcare-associated infections (HAIs) are infections that cause serious mortality and morbidity. This study aimed to investigate the effect of long public holidays on HAIs rates in the intensive care units (ICUs). Methods: The study was conducted in tertiary university education and research hospital, from January 2014 to October 2015. All ICUs are monitored daily by the infection control team by the active surveillance method. In this study, LPH and normal working periods (NWT) that develop HAIs, the bacterial factors that cause HAIs, between periods mortality rates, and overall mortality rates were compared. All data were analyzed with the Epi-Info program (Atlanta, USA) and p-values of <0.05 were considered statistically significant. Results: During the study period, 3082 patients in the ICU were followed up. The HAI rate was 3.5% in NWT and 16.5% in LPH (p=0.001). The examination of bacterial distribution that causes HAIs revealed significantly higher gram-negative bacterial infections in LPH than in NWT [13.7% and 2.4%, respectively (p=0.001)]. The mortality rate examination revealed no significant difference in the overall mortality rates between study periods (p=0.769); infection-related mortality rates were significantly higher in LPH (7.3%; p=0.002). Conclusion: HAIs are affected by LPH. All employees should be motivated to prevent HAIs and should be trained for infection control measures before and after the holidays.