Background: Healthcare-associated infections (HCAIs) pose a significant threat to patient safety and healthcare resources globally, impacting patient safety, treatment outcomes particularly in settings like military hospitals. Aim of the Study: Was to investigate the knowledge and practices of infection control among 15 physicians and 72 nurses at a military fever hospital in Cairo, Egypt. Subjects and methods: A cross-sectional study using a self-administered questionnaire and an observation checklist. The study assessed healthcare providers' knowledge of healthcare-associated infections (HCAIs), causative organisms, transmission routes, infection control measures, and related practices like hand hygiene, personal protective equipment (PPE) use, and safe waste disposal. Results: Significant knowledge deficiencies among physicians; only 33.3% demonstrated satisfactory knowledge, with particularly low scores regarding HCAI definitions (20% satisfactory), isolation procedures (26.7% satisfactory), and safe waste disposal (13.3% satisfactory). Nurses demonstrated comparatively better knowledge, with 65.3% achieving satisfactory scores. Despite these knowledge gaps, both groups exhibited generally adequate practices, with 93.3% of physicians and 94.4% of nurses demonstrating adequate overall practice. However, hand hygiene was a notable area for improvement, with only 20% of physicians demonstrating adequate hand hygiene practices compared to 88.9% of nurses. Statistical analysis indicated that being a nurse and prior training were independent predictors of higher knowledge scores, explaining 29% of the variance. Nursing job alone positively predicted practice scores, explaining 10% of the variance. Conclusion: The study highlights the need for targeted training interventions, particularly for physicians, to address identified knowledge gaps in HCAI definitions, isolation procedures, and safe waste disposal, thereby reinforcing best practices and enhancing patient safety within this unique military healthcare setting.