This study aimed to evaluate the level of knowledge of healthcare workers in Kutahya (a western province of Turkey) about HCV infection and their attitudes toward patients with hepatitis C infection. A total of 335 healthcare workers working in Kutahya Evliya Celebi Research and Training Hospital were included in the study. A questionnaire evaluating demographic characteristics, level of knowledge and attitudes of healthcare workers toward patients with hepatitis C infection was administered to the participants. Thirty-six point four percent of the participants were males. According to the occupations, distributions of the participants were as followings: 54.6% nurse, 25.1% physician, 11.6% trainee nurse, 4.5% intern and 4.2% anesthesia technician. The mean age of healthcare professionals was 30.32 +/- 8.10 years. Fifty-eight point eight percent of them experienced percutaneous injury. The mean knowledge score was 18.05 +/- 3.01 (from a total of 25). There was a statistically significant relationship between knowledge score and age, working year, occupational group (p<0.001). There was also a significant relationship between total attitude score and gender (p=0.006), age (p=0.002), working year (p=0.021) and occupational group (p<0.001). It was determined that as total knowledge score of the participants increased they exhibited a more positive attitude toward patients with hepatitis C infection (p<0.001). A positive relationship was determined between the level of knowledge of healthcare workers and positive attitudes toward patients with hepatitis C infection. Therefore, increasing the level of knowledge is necessary for more positive attitudes.