BackgroundThe transformative potential of artificial intelligence (AI) in higher education is evident, with conversational models like ChatGPT poised to reshape teaching and assessment methods. The rapid evolution of AI models requires a continuous evaluation. AI-based models can offer personalized learning experiences but raises accuracy concerns. MCQs are widely used for competency assessment. The aim of this study was to evaluate ChatGPT performance in medical microbiology MCQs compared to the students’ performance.MethodsThe study employed an 80-MCQ dataset from a 2021 medical microbiology exam at the University of Jordan Doctor of Dental Surgery (DDS) Medical Microbiology 2 course. The exam contained 40 midterm and 40 final MCQs, authored by a single instructor without copyright issues. The MCQs were categorized based on the revised Bloom’s Taxonomy into four categories: Remember, Understand, Analyze, or Evaluate. Metrics, including facility index and discriminative efficiency, were derived from 153 midterm and 154 final exam DDS student performances. ChatGPT 3.5 was used to answer questions, and responses were assessed for correctness and clarity by two independent raters.ResultsChatGPT 3.5 correctly answered 64 out of 80 medical microbiology MCQs (80%) but scored below the student average (80.5/100 vs. 86.21/100). Incorrect ChatGPT responses were more common in MCQs with longer choices (p = 0.025). ChatGPT 3.5 performance varied across cognitive domains: Remember (88.5% correct), Understand (82.4% correct), Analyze (75% correct), Evaluate (72% correct), with no statistically significant differences (p = 0.492). Correct ChatGPT responses received statistically significant higher average clarity and correctness scores compared to incorrect responses.ConclusionThe study findings emphasized the need for ongoing refinement and evaluation of ChatGPT performance. ChatGPT 3.5 showed the potential to correctly and clearly answer medical microbiology MCQs; nevertheless, its performance was below-bar compared to the students. Variability in ChatGPT performance in different cognitive domains should be considered in future studies. The study insights could contribute to the ongoing evaluation of the AI-based models’ role in educational assessment and to augment the traditional methods in higher education.