Background <p>Monkeypox, recently classified as a Public Health Emergency of International Concern by the WHO, presents new challenges for global health systems. As future frontline providers, nursing students play a key role in disease prevention, yet limited knowledge and low health literacy may impair their readiness and heighten anxiety. This study assessed nursing students’ knowledge and anxiety regarding Monkeypox and examined the mediating role of e-health literacy.</p> Methods <p>A cross-sectional study was conducted using an online survey. The sample included 502 nursing students from various Turkish universities who participated between October and December 2024. Data were collected through the Monkeypox Knowledge Form, the Visual Analogue Scale for Anxiety, and the e-Health Literacy Scale. Multivariate linear regression and mediation analysis were performed to assess the relationships among knowledge, anxiety, and e-health literacy.</p> Results <p>The mean Monkeypox knowledge score was 6.89 (SD 3.26), with 52.8% of students demonstrating good knowledge. Anxiety levels were moderate to high, while e-health literacy was at a moderate level. Higher education level (<i>p</i> &lt; 0.001) and e-health literacy <b>(</b><i>p</i> &lt; 0.001<b>)</b> positively predicted Monkeypox knowledge, whereas vaccine hesitancy (<i>p</i> &lt; 0.05) and higher anxiety levels (<i>p</i> &lt; 0.001<b>)</b> were negative predictors. E-health literacy partially mediated the relationship between anxiety and knowledge.</p> Conclusions <p>Findings indicate that e-health literacy significantly influences students’ Mpox-related knowledge and anxiety levels. Enhancing e-health literacy is essential for improving nursing students’ preparedness for public health emergencies. Incorporating health literacy education into nursing curricula and leveraging social media for effective health communication are recommended strategies.</p> Clinical trial number <p>Not applicable.</p>