Background: Pressure ulcers are a critical global health concern. Worldwide, the prevalence of pressure ulcers ranges from 3.4% to 32.4%. In Bangladesh, the prevalence rate is notably high, reaching 43% among bedridden patients. This study aimed to assess the level of nurses' knowledge regarding pressure ulcer prevention at a selected hospital in Barishal, Bangladesh. Methodology: A cross-sectional study was conducted among 103 nurses at Sher-e-Bangla Medical College Hospital, Barishal, using a convenient sampling technique. Data collection was carried out using a 25-item questionnaire, including the Pressure Ulcer Knowledge Assessment Tool 2.0, a revised and validated tool developed at Ghent University, Belgium. The data were analyzed using SPSS software, employing both descriptive and inferential statistics. Results: The average age of the nurses was 35.74 years (SD = 4.51), with more than half (54.4%) being younger than 36 years. Knowledge was assessed on a binary scale (Yes = 1, No = 0), and the mean knowledge score was M = 0.56 (SD = 0.14), indicating poor knowledge of pressure ulcer prevention. Analysis of independent variables revealed that monthly family income (p = 0.04), work experience (p = 0.04), marital status (p = 0.02), professional education level (p = 0.000), and job position (p = 0.02) were significantly associated with nurses' knowledge of pressure ulcer prevention. Conclusion: The study highlights significant knowledge gaps among nurses regarding pressure ulcer prevention in Barishal, Bangladesh. The findings emphasize the need for continuous education and retraining to improve nurses' competence and awareness in pressure ulcer prevention strategies.