Implementation of evidence-based guidelines (EBGs) is an effective measure for prevention of ventilator-associated pneumonia (VAP). Appropriate knowledge, attitude and adherence of healthcare workers (HCWs) to EBGs are necessary factors for implementation of EBGs. This study was conducted with objective of evaluation of knowledge, attitude, and adherence of HCWs to EBGs for prevention of VAP and exploration of the barriers of their implementation in clinical practice. Totally, a total number of 45 HCWs of two pediatric cardiac surgery ICU (PCICUs) participated in this quali-quantitative survey. Knowledge, attitude and adherence of participants was evaluated by a validated multiple-choice questionnaire and barriers of implementation of EBGs was extracted from participants’ answer to an open-ended question of our self-made questionnaire. Knowledge of HCWs was poor and significantly different between nurse assistants (RAs), nurses (RNs), and physicians (MDs) (respectively, 1.25±0.95, 4.53±1.73, and 5.54±2.01, P=0.001). Likewise, attitude of HCWs is not positive and significantly different between NAs, RNs, and MDs (respectively, 32.96±2.42, 34.00±2.44, 36.81±4.35, P=0.003). The adherence of HCWs is not good and different between RAs, RNs, and MDs (respectively, 11.50±1.00, 13.13±1.83, and 17.18±6.06, P=0.17). The Barriers of implementation of EBGs was categorized into four category of individual, organizational, social, and educational factors. Unsatisfying status of knowledge, attitude, and adherence of HCWs is a challenging concern of health-care system, especially in PICUs. In addition to these well-known factors, poor implementation of EBGs is related to many other barriers which should recognized and taken into consideration for designation of infection controlling programs.