COVID-19 was declared a worldwide pandemic in 2020; thus, preventing in-flight infection transmission is important for stopping global spread via air travel. Infection prevention (IP) performance among aircraft cabin crew is crucial for preventing in-flight transmission. We aimed to identify the level of IP performance and factors affecting IP performance among aircraft cabin crew during the COVID-19 pandemic in South Korea. An online survey was conducted with 177 cabin crew members between August and September 2020. The survey assessed IP performance, and IP awareness, using a five-point Likert scale, and also evaluated simulation-based personal protective equipment (PPE) training experience, and organizational culture. The average IP performance score was 4.56 +/- 0.44. Although the performance level for mask-wearing was high (4.73 +/- 0.35), hand hygiene (HH) performance (4.47 +/- 0.56) was low. Multivariate analysis showed that IP performance was significantly associated with IP awareness (p < 0.05) and simulation-based PPE training experience (p < 0.05). Since HH performance was relatively low, cabin crew and airlines should make efforts to improve HH performance. Furthermore, a high level of IP awareness and PPE training experience can improve IP performance among cabin crew members. Therefore, simulation-based PPE training and strategies to improve IP awareness are essential for preventing in-flight infection transmission.