Objectives: The COVID-19 pandemic has disrupted the social life, work environment, and well-being of millions of people. We examined COVID-19’s impact on National Institutes of Health (NIH)–funded extramural principal investigators (PIs) affiliated with public health and preventive medicine departments across the country and their projects; assessed PIs’ confidence in achieving project goals; and investigated the role of age, sex, experience, and team size on PIs’ confidence in achieving project goals during the pandemic. Methods: We sent an anonymous online survey in January 2021 to 1076 extramural PIs affiliated with public health and preventive medicine departments at US institutions; 133 (12.4%) responded. We examined the impact of COVID-19 on the PIs, their project team operations, and their confidence that project objectives would be met, using Likert scales based on age, sex, team size, and PI experience. Results: Of 126 PIs, 94 (74.6%) felt that their day-to-day professional life was impacted a lot or a great deal by COVID-19. More female PIs than male PIs reported that their level of stress changed because of the COVID-19 pandemic. Of 125 PIs, 67 (53.6%) made major adjustments to research operations, 46 (36.8%) made minor adjustments, 5 (4.0%) halted research, and 7 (5.6%) reported not being affected. Of 123 PIs, 89 (72.4%) reported not using NIH COVID-19 accommodations. PIs who led projects 4 or 5 times felt more confident about meeting their research objectives than PIs who led projects 2 or 3 times. Conclusions: Future studies should investigate how to develop more engaging support and communication strategies to assist NIH researchers in mitigating the effects of pandemics or large-scale emergencies.