During the preclinical years, single-best-answer multiple-choice questions (SBA-MCQs) are often used to test the higher-order cognitive processes of medical students (such as application and analysis) while simultaneously assessing lower-order processes (like knowledge and comprehension). Consequently, it can be difficult to pinpoint which learning outcome has been achieved or needs improvement. We developed a new scoring system for SBA-MCQs using a step-by-step methodology to evaluate each learning outcome independently. Enrolled in this study were third-year medical students (n = 316) who had registered in the basic microbiology course at the Faculty of Medicine, Siriraj Hospital, Mahidol University during the academic year 2017. A step-by-step SBA-MCQ with a new scoring system was created and used as a tool to evaluate the validity of the traditional SBA-MCQs that assess two separate outcomes simultaneously. The scores for the two methods, in percentages, were compared using two different questions (SBA-MCQ1 and SBA-MCQ2). SBA-MCQ1 tested the students' knowledge of the causative agent of a specific infectious disease and the basic characteristics of the microorganism, while SBA-MCQ2 tested their knowledge of the causative agent of a specific infectious disease and the pathogenic mechanism of the microorganism. The mean score obtained with the traditional SBA-MCQs was significantly lower than that obtained with the step-by-step SBA-MCQs (85.9% for the traditional approach versus 90.9% for step-by-step SBA-MCQ1; p < 0.001; and 81.5% for the traditional system versus 87.4% for step-by-step SBA-MCQ2; p < 0.001). Moreover, 65.8% and 87.8% of the students scored lower with the traditional SBA-MCQ1 and the traditional SBA-MCQ2, respectively, than with the corresponding sets of step-by-step SBA-MCQ questions. These results suggest that traditional SBA-MCQ scores need to be interpreted with caution because they have the potential to underestimate the learning achievement of students. Therefore, the step-by-step SBA-MCQ is preferable to the traditional SBA-MCQs and is recommended for use in examinations during the preclinical years.