Background: Compliance with the evidence-based isolation precaution guidelines is essential to prevent the spread of infectious diseases among hospitalized patients and its ensuing complications. Objectives: This study aimed to assess the nurses' knowledge towards the infectious diseases’ isolation precautions in governmental tertiary care hospitals and to identify the relationship between nurse's characteristics concerning their knowledge and practices. Method: A cross-sectional study was conducted in ten tertiary care hospitals using a self-administrated questionnaire to collect nurses' characteristics and to assess their knowledge level. Nurses' practices were evaluated using an observation checklist Univariate, bivariate, and logistic regression analysis were applied. Results: The study revealed that 35.8% of nurses had a good knowledge regarding isolation precautions and 41.6% had good practices score. Some demographic variables inclined Nurses' knowledge; age, gender, education, and experience. While their practice score was affected by; gender, work experience, and unit of work (ICU vs. non- ICU). A statistically significant positive correlation was revealed between the overall knowledge score and the overall practice score of nurses regarding adherence to the infectious diseases isolation precautions(r=0.3). Participants' education and duration of working experience were the significant predictors for higher knowledge scores (p <0.05). Conclusion: The tailored multi-modal training approach can improve nurses' knowledge and promote their experiences towards isolation precautions