Background: The World Health Organization (WHO) verified 780 cases of monkeypox across 27 countries between 13 May 2022 and 2 June 2022. The aim of our study was to assess the level of awareness of human monkeypox virus among Syrian medical students, general practitioners, medical residents, and specialists. Methods: A cross-sectional online survey was performed in Syria between May 2 and September 8, 2022. The survey consisted of 53 questions within the following three categories: demographic information, work-related details, and monkeypox knowledge. Results: In total, 1257 Syrian healthcare workers and medical students were enrolled in our study. The animal host and incubation time for monkeypox were correctly identified by just 2.7% and 33.3% of responders, respectively. Sixty percent of the study sample thought that the symptoms of monkeypox and smallpox are identical. No statistically significant associations were found between predictor variables and knowledge regarding monkeypox (p-value > 0.05). Conclusion: Education and awareness regarding monkeypox vaccinations are of paramount importance. It is essential that clinical doctors are adequately aware of this disease, in order to avoid an uncontrolled situation, as experienced with COVID-19.