Abstract Background Antibiotic overuse causes antibiotic resistance, one of the most important threats to human health. Older adults, particularly those in nursing homes, often receive antibiotics when they are not indicated. Methods To understand knowledge, attitudes, and behaviors of nursing home (NH) nurses and community-dwelling older adults towards antibiotic use, especially in clinical situations consistent with antibiotic overuse, we conducted a mixed-method survey in two NHs and one Family Medicine clinic in North Carolina, among English-speaking nurses and community-dwelling, cognitively intact adults aged 65 years or older. Based on the Knowledge-Attitude-Practice model, the survey assessed knowledge, attitudes, and behavior towards antibiotic use, including three vignettes designed to elicit possible antibiotic overuse: asymptomatic bacteriuria (ASB), a viral upper respiratory illness (URI), and a wound from a fall. Results Of 31 NH nurses and 66 community-dwelling older adults, 70% reported knowledge of the dangers of taking antibiotics. Nurses more often reported evidence-based attitudes towards antibiotics than older adults, except 39% agreed with the statement “by the time I am sick enough to go to the doctor with a cold, I expect an antibiotic”, while only 28% of older adults agreed with it. A majority of nurses did not see the need for antibiotics in any of the three vignettes: 77% for the ASB vignette, 87% for the URI vignette, and 97% for the wound vignette. Among older adults, 50% did not perceive a need for antibiotics in the ASB vignette, 58% in the URI vignette, and 74% in the wound vignette. Conclusions While a substantial minority had no knowledge of the dangers of antibiotic use, non-evidence-based attitudes towards antibiotics, and behaviors indicating inappropriate management of suspected infections, most NH nurses and community-dwelling older adults know the harms of antibiotic use and demonstrate evidence-based attitudes and behaviors. However, more work is needed to improve the knowledge, attitudes and behaviors that may contribute to antibiotic overuse.