Background: Breast cancer is a significant global health concern, with its incidence steadily rising in both developed and developing nations. It is the leading cause of cancer-related deaths among women, accounting for 16% of cancer fatalities in adult females. This study aimed to assess the level of breast cancer knowledge among nurses working at a tertiary-level hospital in Barishal. Methodology: A cross-sectional study was conducted among 266 nurses at Sher-E-Bangla Medical College Hospital, Barishal. A convenient sampling technique was applied, and data were collected using a self-structured questionnaire developed through an extensive literature review. Data analysis was performed using SPSS-23, employing both descriptive and inferential statistics. Results: Nearly 97% of nurses recognized breast cancer as a life-threatening disease, while more than half (61%) disagreed with the notion that breast cancer is preventable. The overall mean knowledge score on breast cancer was 3.75 (SD = 0.40), indicating an average to moderate level of knowledge. Marital status (t = -2.84, p<0.05) was found to have a statistically significant association with breast cancer knowledge, with married nurses demonstrating higher awareness than their single counterparts. Additionally, significant differences in knowledge levels were observed between Muslim and non-Muslim nurses, with non-Muslim nurses exhibiting greater awareness of breast cancer. Conclusion: The findings of this study provide valuable insights into the level of breast cancer knowledge among nurses at a tertiary-level hospital in Barishal. The results can serve as a guide for improving nurses' understanding of breast cancer and developing strategies to reduce associated risks. Furthermore, these findings emphasize the need for health education programs aimed at raising awareness and disseminating crucial information on breast cancer prevention and early detection.