Nosocomial infections are a major source of morbidity and mortality, affecting more than 2 million patients annually in the United States. Furthermore, the hospital environment supporting the acquisition of resistance to antibiotic agents by pathogens, complicates the treatment of infections due to drug-resistance of these pathogens. Ethnopharmacological reports support the use of neem (Azadirachta indica) against bacterial and fungal infections such as typhoid, yeast infection, and periodontitis. However, there is a lack of research about the effect of neem specifically on nosocomial organisms. We conducted this study to evaluate the effect of aqueous and ethanolic extracts from neem leaves and neem oil on the growth of several human pathogens which are known to cause hospital-acquired, or nosocomial, infections, including Saccharomyces cerevisiae, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Neem extract in distilled water showed the strongest average inhibition across all microorganisms except S. aureus. Activity of neem extract in 95% ethanol was comparable to that of 10% bleach. Under the conditions of this study, we concluded that neem leaf extract has a significant antimicrobial effect against nosocomial organisms, supporting its use as an alternative or combination treatment for hospital-acquired infections.