Aim: The aim of this study is to assess knowledge levels of intensive care unit staff working at four training and research hospitals in Ankara about infection prevention measures before and after receiving training, and to determine the factors which will affect knowledge levels about infection prevention measures. Material and Methods: Doctors and nurses who work at intensive care units in Ankara Numune Training and Research Hospital (TRH), Ankara TRH, Dr. Zekai Tahir Burak Women’s Health TRH and Ankara Turkey High Speciality TRH participated voluntarily in the study between March-April 2015. A questionnaire comprising ten questions about infection prevention measures was administered to healthcare professionals who volunteered to participate in the study (doctors and nurses), and then participants received a training regarding infection prevention measures, after the training, the same questionnaire was administered in order to evaluate the differences statistically. One hundred-sixty four health care workers were included in the study. Answers given to the questionnaire items before and after the training were listed in Excel program and statistical analysis was carried out with SPSS 15.0 (Statistical Package for the Social Sciences) data analysis program. Value of p≤0.05 was considered as statistically significant. Results: While the success rate was 72% before the training, it reached to 83% after the training, the average number of correct answers was eight before the training (1-10), after the training, the average number of correct answers was nine (4-10) and statistically significant difference was observed (p=0.00). Knowledge levels of health care professionals about infection prevention measures significantly increased after the training. Those who have received such training in their earlier professional life had statistically higher knowledge levels compared to those who have not received such training earlier in their career. It was observed that knowledge level increased through training in both genders and in every age group. Conclusion: As a result of the renewal of knowledge with continuous training of health personnel and updating services as well as evaluating the training by using the tests before and after the training is an effective approach to the prevention of infections associated with healthcare associated infections.