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Introduction: Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause frequent outbreaks in hospitals in Saudi Arabia. Since healthcare workers (HCWs) have a higher risk of acquiring and spreading MERS-CoV, we aimed to evaluate the perceived risk and anxiety level of HCWs in Saudi Arabia regarding MERS. Methodology: An anonymous, self-administered questionnaire was sent online to HCWs at King Khalid University Hospital in Saudi Arabia. The total knowledge and anxiety scores were calculated. Logistic regression analyses were used to identify predictors of high anxiety scores. Results: Of 591 (70%) HCWs that responded, 284 (55%), 164 (32%), and 68 (13.2%) were physicians, nurses, and technicians, respectively. Physicians obtained a lower median knowledge score (6/9) compared to other professions (7/9). The mean anxiety score was similar for physicians and other HCWs (3/5); however, non-physicians expressed higher levels of anxiety toward the risk of transmitting MERS-CoV to their families, with an anxiety score of 4/5. The ability of the virus to cause severe disease or death was the most frequently reported reason for worry by physicians (89.7%) and non-physicians (87.2%). Overall, 80% of physicians and 90% of non-physicians reported improvement in adherence to hand hygiene and standard precautions while in hospital (p = 0.002). Concern over transmitting MERS-CoV to family members was the most predictive factor for anxiety among non-physician HCWs. Conclusion: A significant proportion of HCWs expressed anxiety about the risk of acquiring MERS-CoV infection. Healthcare institutions need to develop an integrated psychological response for HCWs to the occupational and psychological challenge of MERS-CoV outbreaks.