Background <p>Hepatitis B infection (HBI) is a healthcare-associated bloodborne infection that has a considerable impact on healthcare workers, including undergraduate nurses, during their clinical training. Appropriate knowledge, attitudes, and practices (KAP) are essential to minimize HBI. Therefore, this study aimed to investigate the KAP of nursing undergraduates regarding HBI.</p> Methods <p>A descriptive cross-sectional study was conducted among 217 nursing undergraduates of General Sir John Kotelawala Defence University in Sri Lanka. A pre-validated, self-administered questionnaire was used to collect data. Socio-demographics, and KAP related to HBI prevention were assessed. The Spearman correlation and multinomial logistic regression was used to analyze associations between considered factors.</p> Results <p>The majority of respondents exhibited moderate knowledge (57.6%), attitudes (59.0%), and practices (68.7%) towards HBI. Significant associations were observed between study year, and mean scores of knowledge (<i>P</i> = 0.022) and practices (<i>P</i> = 0.000), while age was significantly associated with practice scores (<i>P</i> = 0.001). Multivariate logistic regression analysis revealed that respondents with high knowledge levels were approximately two times more likely to engage in good practices (aOR = 2.422, <i>P</i> = 0.000). Whilst, participants with moderate attitudes were twice likely to demonstrate good practices (aOR = 2.478, <i>P</i> = 0.000). The level of attitudes showed a significant weak positive correlation with practices of HBI (<i>r</i> = 0.311, <i>p</i> &lt; 0.001).</p> Conclusions <p>KAP of HBI are crucial among nursing undergraduates. Targeted training programs at the beginning of clinical training and adhering to infection control protocols are needed in clinical settings to improve awareness and reduce the risk on HBI.</p>