Background: There is a shift towards increased use of telemedicine applications for healthcare service provision and delivery. Thus, awareness among healthcare practitioners of telemedicine policies is critical for proper implementation and utilization of telemedicine technology.Objective: This study assesses the level of computer access and literacy, knowledge of telemedicine policies and technology, perceptions, and willingness to use telemedicine among healthcare practitioners working in ambulatory care clinics. Methods: An observational cross-sectional study was conducted at King Fahad Armed Forces Hospital in Jeddah. Data were collected between February and March 2021 using a self-completed online survey. Results: Of 136 healthcare practitioners surveyed, we found that over half had average to high knowledge about telemedicine technology, tools, guidelines, security, and privacy policies within the hospital. 95% were willing to use telemedicine to consult with large centers in their medical or clinical specialty and support further implementation of telemedicine technology in the hospital. 90% expressed a need for continuous training in the use of telemedicine. Conclusion:The current study shows that there is inadequate computer access and knowledge of telemedicine, but there were very positive perceptions and willingness related to telemedicine among healthcare practitioners working in the ambulatory care clinics. There is an urgent need for orientation and training programs that focus on the technology and applications of telemedicine, as well as current policies.