This study explores students' emotions in the undergraduate chemistry laboratory, focusing particularly on emotional responses to making mistakes. Using an ecological momentary assessment design, we collected on-task data from 240 students and 1635 learning situations during the laboratory sessions. A multilevel structural equation modeling framework, including a model with random slopes, was used to examine how situational factors (i.e., type of mistake and support in solving the mistake) and individual factor (i.e., gender, performance level, and subjective task values) affected students' emotional responses. Our findings suggest that mistakes typically trigger negative activating emotions in students. Individual factors also influenced emotional responses—well-performing students were less prone to elevated anxiety and frustration, remaining more relaxed in mistake situations, whereas students with high perceived emotional costs of laboratory activities experienced a more intense increase in anxiety in mistake situations. The type of mistake played an important role: careless mistakes were associated with more positive emotions, whereas students experienced increased anxiety and frustration and decreased positive emotions when the mistake stemmed from skill or knowledge gaps. The form of support also mattered: teacher support was linked to intensified anxiety and frustration in students, while peer support showed no statistically significant impacts on emotional levels. To foster meaningful learning, targeted support in emotional regulation should be provided for lower-performing students and those with high perceived emotional costs in mistake situations, particularly when mistakes stem from a lack of skill or knowledge.