Biofilms formed by K. pneumoniae are more important for their role in antibiotic resistance and chronic infections. The present study focuses on comparing the efficacy of Azadirachta indica (Neem) extract on the biofilm formation of K. pneumoniae on the wall surface of different substrates. The development of biofilm-forming ability of K. pneumoniae was studied in the presence of Neem extract concentrations. Accordingly, a high increase in the concentration of Neem extract resulted in a significant decrease in biofilm formation. The present study confirms the potential effect of A. indica for application as a natural and eco-friendly biofilm control agent, particularly against K. pneumoniae in environmental settings. Based on these premises, further research into the applications of Neem extracts in biofilm management opens up new perspectives related to the inclusion of Neem extract in antimicrobial treatments against biofilm infections.