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Fear of negative evaluation (FNE), defined as a sense of dread associated with being negatively judged in a social situation, has been identified as the primary factor underlying undergraduate anxiety in active-learning science courses. However, no quantitative studies have examined the extent to which science undergraduates experience FNE and how they are impacted by FNE in college science courses. To address this gap, we surveyed 566 undergraduates from one university in the U.S. Southwest who were enrolled in life sciences courses where they had opportunities to speak in front of the whole class. Participants were asked a suite of questions regarding their experiences with FNE in large-enrollment college science courses. We found that first-generation college students, LGBTQ+ students, and students with disabilities reported disproportionately high levels of FNE compared with their counterparts. Additionally, students reported that FNE can cause them to overthink their responses and participate less in class. Participants rated being cold called and presenting alone as forms of whole-class participation that elicit the highest levels of FNE. This research highlights the impact of FNE on undergraduates and provides student-generated recommendations to reduce FNE in active-learning science courses.