This study investigates the role of mistakes and affective experiences during online pre-lab activities in predicting students’ situational engagement (conceptualized here as a simultaneous experience of interest, skill, and challenge, i.e. optimal learning moments) in subsequent laboratory sessions in an undergraduate chemistry laboratory course (n=256). The data collection followed an ecological momentary assessment design. We specified multilevel structural equation models (MSEM), including two- and three-level structural equation models, to examine how mistakes impacted students’ situational engagement during pre-lab activities and subsequent laboratory sessions. The findings indicate that mistakes in pre-lab tasks were associated with lower perceived skill and higher experience of challenge during that task, but did not predict students’ interest, skill, challenge, or situational engagement in the subsequent laboratory session. Autoregressive effects from pre-lab activities to students’ situational engagement during lab sessions were observed across all elements of engagement, while skill and challenge during pre-lab activities also predicted higher interest in the subsequent laboratory session. Based on our findings, we propose that mistakes in the pre-lab activities do not play a significant role in predicting students’ engagement upon entering the laboratory, affective experiences during pre-lab activities can play a significant role in predicting students’ engagement in the laboratory, and that laboratory engagement could be enhanced by providing students with sufficiently challenging pre-lab activities.