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<b>Introduction:</b> Knowledge and attitudes towards proper antibiotic usage among students in medical and other health allied fields play a vital role in limiting the pandemic of antibiotic resistance. This study aimed to assess knowledge and attitudes toward antibiotic usage among pre-professional students at King Saud bin Abdulaziz University for Health Sciences on Jeddah Campus, Saudi Arabia. <b>Methods:</b> A self-administered cross-sectional survey of 347 first year students was conducted at the College of Science and Health Professions, King Saud bin Abdulaziz University for Health Sciences using a validated questionnaire. <b>Results:</b> Nearly 63% of the students had a moderate level of knowledge. Two third of the students (69.7%) correctly knew that antibiotics are indicated for the treatment of bacterial infections. However, about 36% of the students incorrectly thought that antibiotics are also used to treat viral infections, while 28.5% were not sure. Only 38.8% of the students were aware of antibiotic resistance phenomena in relation to the overuse of antibiotics. Furthermore, only 27.3% correctly knew that penicillin is an antibiotic, and 74.4% were not sure if Paracetamol is an antibiotic. With regard to attitudes, 25.7% believed that taking antibiotics when having cold symptoms could help them to recover faster, while 39.6% expected antibiotics to be prescribed for common cold symptoms. <b>Conclusion:</b> The students have misconception regarding antibiotic use indications. Therefore, awareness campaigns are needed to promote student&#8217;s use of antibiotics in young generations particularly among the pre-professional health sciences students.