Background: Globally, the need to address strategies for preventing infections associated with health care has increased worldwide. In the city of Valledupar, Colombia, reports of bacteria resistant to chemical or enzymatic biocides in hospital environments and surfaces are increasingly frequent, evidencing the importance of conducting studies aimed at identifying alternative active ingredients for disinfectant products. Objective: Evaluate the in vitro effectiveness of Neem leaves extract over bacteria strains isolated from different areas and surfaces of a health institution in Valledupar, compared to disinfectants for hospital use, an enzymatic detergent, and a commercial chemical disinfectant. Methods: Biocidal activities on bacteria isolated from hospital surfaces, such as Acinetobacter baumanni, Bacillus subtilis, Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus sp, and Stenotrophomonas malthophila were analyzed. The Neem leaves extract was evaluated at concentrations of 3, 4, and 5 % for each bacterium during 15-minute contact time, incubated at 37 °C for 18 hours. We compared two antimicrobial chemicals, a disinfectant (based on formaldehyde, cetrimide, and glutaraldehyde), and an enzymatic detergent (based on protease, lipase, and amylase). Results: The aqueous Neem extract did not show significant differences with the other treatments with 99.48 to 100% inhibition against bacteria of the species Acinetobacter baumanni, Enterobacter aerogenes, Staphylococcus aureus, and Micrococcus sp.; Bacillus subtilis, and Stenotrophomonas maltophilia strains were the most resistant strains inhibited by enzymatic detergent and disinfectant, respectively. None of the products evaluated were effective against all in vitrostrains. Conclusions: These data show Neem’s bacteriostatic properties, its potential in in-hospital products, and the need to combine different active ingredients in a disinfection plan.