Healthcare-associated infections have become a serious public health problem. Various types of information systems have begun to be applied in hospital infection prevention and control (IPC) practice. Clinicians are the key users of these systems, but few studies have assessed the use of infection prevention and control information systems (IPCISs) from their perspective.This study aimed to (1) apply the extended DeLone and McLean Information Systems Success model (D&M model) that incorporates IPC culture to examine how technical factors like information quality, system quality, and service quality, as well as organizational culture factors affect clinicians' use intention, satisfaction, and perceived net benefits, and (2) identify which factors are the most important for clinicians' use intention.A total of 12,317 clinicians from secondary and tertiary hospitals were surveyed online. Data were analyzed using partial least squares-structural equation modeling and the importance-performance matrix analysis.Among the technical factors, system quality (β=.089-.252; P<.001), information quality (β=.294-.102; P<.001), and service quality (β=.126-.411; P<.001) were significantly related to user satisfaction (R2=0.833), use intention (R2=0.821), and perceived net benefits (communication benefits [R2=0.676], decision-making benefits [R2=0.624], and organizational benefits [R2=0.656]). IPC culture had an effect on use intention (β=.059; P<.001), and it also indirectly affected perceived net benefits (β=.461-.474; P<.001). In the importance-performance matrix analysis, the attributes of service quality (providing user training) and information quality (readability) were present in the fourth quadrant, indicating their high importance and low performance.This study provides valuable insights into IPCIS usage among clinicians from the perspectives of technology and organization culture factors. It found that technical factors (system quality, information quality, and service quality) and hospital IPC culture have an impact on the successful use of IPCISs after evaluating the application of IPCISs based on the extended D&M model. Furthermore, service quality and information quality showed higher importance and lower performance for use intention. These findings provide empirical evidence and specific practical directions for further improving the construction of IPCISs.©Feiyang Zheng, Kang Wang, Qianning Wang, Tiantian Yu, Lu Wang, Xinping Zhang, Xiang Wu, Qian Zhou, Li Tan. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 22.06.2023.