Nosocomial infections negatively affect the results obtained from healthcare services. Healthcare-associated infections (HAIs) are most common in patients in intensive care units (ICUs) and immunosuppressed patients. The World Health Organization estimates that HAIs affect hundreds of millions of patients each year, causing major economic losses. Many national health organizations continue to implement precautions to reduce the incidence of hospital infections. Many studies have been conducted on the contamination of frequently used surfaces in hospitals with various microorganisms and their role in causing HAIs. Bacillus subtilis (B. subtilis) is a Gram-positive bacillus. The literature reports that bacillus species can adhere to surfaces through their spore structures. It has been noted that they can survive for approximately four weeks in environments without food, can be transmitted to patients from contaminated surfaces, and can cause infection depending on the number of spores. In numerous studies, B. subtilis has been isolated from various samples including blood, cerebrospinal fluid, liver and spleen abscesses, and vitreous fluid. It is important to recognize that these bacteria may be not only contaminants but also pathogenic microorganisms that can cause HAIs. Understanding the correlation between B. subtilis and HAI will significantly benefit healthcare services.