Hospital-acquired infections (HAIs) are infections which are absent at the time of admission and would have been acquired after hospitalization. The purpose of the study was to evaluate the knowledge and perception levels on preventing HAI among health care professionals in tertiary care setup. The semi-structured tool was used for data collection. Out of 367 health care professionals, 262 participants were included in the study. Data were analyzed with descriptive and inferential statistics. The mean level of knowledge and perception score on HAI was 19.58 ± 2.70 and 51.97 ± 5.62. There was a positive correlation among the level of knowledge with level of perception on the prevention of HAI (r=0.16) at p>0.01. The knowledge and perception level was significantly associated with few demographic variables such as following protocols of hand hygiene,trainings on HAI within last year and functioning infection control department in their institution at p = 0.001. Health care professionals have adequate knowledge and perception regarding the prevention of HAI. There should be emphasized refreshment and updates on the current trends on the prevention of HAI among the health care professionals through intensive education and conducting training courses.