Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an emerging pathogen with a pandemic potential and a higher burden in Saudi Arabia. This cross-sectional study aimed to investigate the knowledge and attitude toward MERS-CoV among Health Colleges’ students in a Saudi public university. The study included 540 students. A well-constructed questionnaire containing 14 knowledge and 8 attitude items was completed by all participants. Overall, >80% of students were aware about MERS-CoV aetiology, mode of transmission, risk factors and signs and symptoms. Knowledge scores revealed that 42% of participants have sufficient knowledge. Medical students had significantly better knowledge compared to students in other Health Colleges (p = 0.001). Concerning attitude, >50% of students exhibited a positive attitude toward MERS-CoV. This study showed a good knowledge level and a strong positive attitude towards protection from MERS-CoV among Health Colleges’ students. However, there is a noticeable defect in the knowledge level about the microbiology of the virus and the infection control measures. The findings of this study highlight the need to design educational courses on new emerging diseases, field epidemiology, infection control practice and public health to all Health Colleges’ students to prepare them for dealing with these types of health emergencies in their future healthcare practice. Besides, continuous awareness campaigns about new emerging diseases are warranted for all Health Colleges’ students to improve their knowledge and attitude toward emerging infection trends.