Self-efficacy has a strong influence on the learning and motivation of science students at the postsecondary level, especially in upper division science classes, which are key to student success in science majors. This empirical mixed methods research study (N = 205) examines the associations between students' participation in an online preparation course and student self-efficacy in organic chemistry. Qualitative content analysis indicated that students benefited from the online preparatory course in the subsequent organic chemistry course series. The analysis of students' clickstream data indicated that students with self-efficacy ratings in the top 10th percentile exhibited more frequent and consistent engagement with relevant course materials compared to students in the bottom 10th percentile. Notably, linear regression models indicated that participation in the online preparatory course was associated with higher long-term self-efficacy for first-generation college students. These results suggest that online preparatory courses may benefit some students' self-efficacy in demanding science courses.