This study evaluated the knowledge and anxiety levels of nurses in Türkiye regarding human monkeypox virus (Mpox) infection and explored related factors such as institutional training and preparedness. A descriptive, cross-sectional study was conducted between October 2024 and January 2025 with 214 actively employed nurses. Data were collected using a socio-demographic questionnaire, a nurse introduction form, a form assessing thoughts about Mpox, a knowledge assessment form, and the Epidemic Anxiety Scale (EAS). The mean epidemic anxiety score was 48.36 ± 19.26, with significantly higher anxiety levels among female nurses. Greater Mpox knowledge was associated with higher job satisfaction, access to institutional information, and participation in training. Fear of infection, fear of transmitting the virus, and fear of losing loved ones significantly influenced knowledge levels. Each unit increase in perceived information adequacy was associated with a 6.56-point rise in knowledge scores. The findings reveal a substantial knowledge gap about Mpox among nurses in Türkiye, which may hinder outbreak preparedness. Strengthening educational initiatives and integrating epidemic management into nursing curricula are essential to improving nurses' readiness and protecting public health.