Oral hygiene has a vital role on patient comfort, well-being and general health as it is a critical and basic nursing procedure. Morbidity, mortality, hospital stays and health care costs are increased due to poor oral hygiene in hospitalized patients. Nurses are responsible for rendering oral care to patients. However, they usually undermine and perform it sub-standardly due to many reasons, one of which is having inadequate level of knowledge. The aim of this study was, therefore, to assess nurses‟ level of knowledge on oral care. Methods: A descriptive cross-sectional study was conducted at Orotta medical-surgical national referral hospital (OMSNRH). Data was collected by interviewing all (n=73) diploma and associate nurses using pretested and structured questionnaire. Independent samples t-test and analysis of variance (ANOVA) were performed to analyze the data using SPSS (Version 22). Results: A total of 73 participants were enrolled, out of them 56.2% were diploma nurses. 97.3% of the participants had been thought about oral care but only 53.5% practiced during their education. The majority (63.0%) of the participants had correctly answered the need for assessment of oral cavity within 24 hours of admission. Besides, 41.1% of the participants stated tooth brush as the most effective method of oral care. However, 91.8% of the participants had poor knowledge score. At bivariate level, knowledge score was significantly different across the categories of educational level (p=0.003), and working ward (p=0.001). At multivariable level, only working ward (p=0.025) was found to be a significant determinant of the knowledge score. Working ward was found to have medium effect in determining the knowledge score (Cohen‟s f=0.21). Conclusions: A substantial gap in knowledge among the participants was observed. Therefore, further in-service training, and preparation and implementation of standardized protocol to promote the oral health of patients are promptly recommended.