Hospital-acquired infections (HAIs), also known as nosocomial infections, are infections acquired in healthcare settings that were neither present nor incubating at the time of admission. These infections represent a critical challenge globally, significantly increasing patient morbidity, mortality, duration of hospital stays, and healthcare costs—particularly in intensive care units and among vulnerable populations. This entry comprehensively explores the multifaceted dimensions of HAIs, including their epidemiological burden, common types (such as bloodstream infections, surgical site infections, catheter-associated urinary tract infections, and ventilator-associated pneumonia), and the major microbial culprits, including multidrug-resistant organisms like methicillin-resistant Staphylococcus aureus, vancomycin Enterococci species, and broad-spectrum beta-lactamases-producing Gram-negative bacilli. It also highlights key risk factors such as immunosuppression, device use, and poor infection control practices. Mechanisms of transmission, ranging from contact to airborne and droplet spread, are discussed in depth. Emphasis is placed on advances in diagnostics (polymerase chain reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, artificial intelligence-based systems), surveillance methodologies, and evidence-based infection prevention protocols, including hand hygiene, personal protective equipment, and device-care bundles. This entry concludes by addressing current challenges, such as antimicrobial resistance and the need for inclusive, technologically integrated, and globally coordinated strategies to reduce the HAI burden. By integrating recent evidence and World Health Organization guidelines, this entry serves as a practical and scientific reference for clinicians, microbiologists, infection control teams, and public health policymakers.