Background: Healthcare-associated infections (HAIs) remain a major public health challenge worldwide, contributing significantly to patient morbidity, mortality, prolonged hospitalization, and increased healthcare costs. Nurses play a central role in the implementation of Infection Prevention and Control (IPC) measures; therefore, adequate awareness of IPC principles is essential for ensuring patient safety and reducing infection transmission within healthcare facilities. Objective: This study assessed the level of awareness of Infection Prevention and Control among nurses at a tertiary healthcare institution in Southern Nigeria and identified existing knowledge gaps that may hinder effective IPC implementation. Materials and Methods: A descriptive cross-sectional survey was conducted among 197 registered nurses at the Federal Medical Centre, Yenagoa, Bayelsa State, Nigeria. Participants were selected using a simple random sampling technique. Data were collected using a structured self-administered questionnaire comprising socio-demographic variables and ten awareness-related items. Responses were measured on a five-point Likert scale. Data were analyzed using descriptive statistics, including frequencies, percentages, means, and standard deviations. A mean score of 3.0 and above was considered indicative of good awareness. Results: The overall awareness of IPC among nurses was high, with a global mean score of 3.76 ± 1.05. The highest awareness was recorded for the importance of hand hygiene in preventing infections (4.32 ± 0.78), familiarity with the proper use of personal protective equipment (4.09 ± 0.93), and understanding the rationale behind regular IPC training (4.04 ± 0.92). However, lower awareness scores were observed regarding adequacy of IPC training received (3.43 ± 1.14), being up-to-date with current IPC guidelines (3.43 ± 1.18), and regular review of IPC protocols (3.43 ± 1.18). Overall, 148 (75.2%) nurses demonstrated good awareness, while 49 (24.8%) had poor awareness. Conclusion: Nurses demonstrated good overall awareness of Infection Prevention and Control measures. Nevertheless, important gaps exist in continuous professional development, access to updated IPC guidelines, and routine review of institutional protocols. Strengthening regular training and dissemination of current IPC information may further improve awareness and support effective infection prevention practices.