Biofilm represents a potential strut in bacterial treatment failure. It has a dual action; it affords microbial resistance against antibiotics and facilitate transmission of pathogenic bacteria. Nosocomial bacteria pose a serious problem in healthcare units; it prolongs patient hospital stay and increases the mortality rates beside other awful economical effect. This study was planned for targeting nosocomial bacterial biofilm using natural and biologically safe compounds like Chitosan and/or Pluronic F127. Ninety-five isolates were recovered from 107 nosocomial clinical samples. Different bacterial and fungal species were detected, from which Klebsiella pneumonia (23%), Pseudomonas aeruginosa (19%), Acinetobacter baumannii (18%) and E.coli (17%) were the predominate organisms. Pseudomonas aeruginosa, Acinetobacter baumanni and Klebsiella pneumonia were the abundant antibiotic resistant strains with multi-resistance pattern of 72%, 65% and 59%, respectively. A significant percentage of these isolates were strong biofilm forming. Herein, we investigate the effect of Chitosan and Pluronic F127 alone and in combination with each other against biofilm production. Chitosan show variable degree of biofilm inhibition, while Pluronic F127 was able to retard biofilm formation by 80% to 90% in most strain. There is no significant difference (P 0.05) between Pluronic F127 alone and its effect in combination with Chitosan.