Background: Coronavirus disease 2019 (COVID-19) has been recognized as a pandemic by the World Health Organization (WHO). Inadequate knowledge and lack of practice of preventive measures of the non-teaching Staff have a direct influence on students and teachers, as students and teachers have frequent interaction with the Non-Teaching Staff. This study aimed to assess the knowledge and practice of preventive measures against Covid-19 among the non-teaching Staff of Ayub Medical College. Methods: A cross-sectional study was conducted at Ayub Medical College between 1st March and 31st October 2021. The study population was a non-teaching staff of Ayub medical college. A structured paper questionnaire was developed after the review of the literature. Data were analyzed using the statistical package for social sciences SPSS version 26. Results: The residence-based distribution shows both urban and rural populations have good knowledge and satisfactory practice score, with an overall KP score more among urban residents. Results also show that participants who had an education level of intermediate or above have higher KP scores as compared to less educated participants. Conclusion: Our survey revealed that most non-teaching staff has adequate knowledge about COVID and they are also practising preventive measures. Knowledge and practice are more among males, urban residents and the literate population.