Graduate students in the United States are reporting increased anxiety, affecting their mental health and attrition in graduate programs. Yet we are only beginning to understand what contributes to graduate student anxiety. Biology Graduate Teaching Assistants (GTAs) have simultaneous roles as teachers, researchers, students, and employees, and factors associated with these tasks may contribute to anxieties in graduate school, particularly in relation to teaching and research responsibilities. To explore factors related to GTA teaching and research anxieties, and guided by social cognitive career theory, we interviewed 23 Biology GTAs at a research-intensive southeastern university. Thematic analysis of interview transcripts revealed five major factors related to GTA anxieties: negative impact on self, negative impact on others, lack of self-efficacy, role tension, and personal anxieties. Lack of self-efficacy was most prevalent for research anxieties, compared to teaching anxieties, where the impact on others (e.g. students) was most prevalent. In research contexts, GTAs with academic career aspirations expressed less anxiety about role tensions compared to GTAs with non-academic career goals. By investigating GTA anxieties, this work can inform professional development or mental health interventions for GTAs and encourage greater awareness and dialogue about mental health issues in academia.