Background Since glaucoma-related blindness may only be prevented with early identification and treatment, finding asymptomatic individuals in the target population is critical. It is thus important to determine the awareness and knowledge on glaucoma. Objective To determine the knowledge and awareness on glaucoma among the staff of a tertiary level hospital in central Nepal. Method This was a cross sectional study among the staffs of Dhulikhel Hospital who participated voluntarily in the study. The data was collected through the administration of semi-structured questionnaires. R version 4.0.3 (2020-10-10) was used for the data analysis. Categorical variables are presented as frequency (percentages) and the numerical ones are presented as mean (standard deviation). Adjusted odds ratio (AOR) with 95% confidence interval were estimated through logistic regression analysis for determining factors associated with awareness and knowledge of glaucoma. Result A total of 107 (89.2%) participants had heard about glaucoma of which 93 (86.9%) had good knowledge and 14(13.1%) had poor knowledge on glaucoma. The mean score of knowledge was 64.1±16.9. Only 47.7% of the participants said that glaucoma also has an asymptomatic course. We could not find an association of awareness and knowledge of glaucoma with any of the selected sociodemographic variables. Conclusion Although there was a fairly good glaucoma related awareness and knowledge among hospital staff, we found lack of understanding on some critical issues on glaucoma, mainly related to its asymptomatic nature. There is thus room for improvement in educating about glaucoma even in healthcare settings. Given the necessity of expanding glaucoma awareness in the public, hospital staff might potentially serve as messengers to the communities.