This study aims to evaluate the effect of lipopeptide carboxymethyl chitosan nanoparticles on Staphylococcus aureus biofilm as part of the development of a new anti-biofilm material. The study had three stages. Firstly, we assessed the Staphylococcus aureus capability to form biofilm and enumerated the number of attached bacteria and free bacteria; secondly, we determined the inhibitory effect of different concentrations of Bacillus natto antimicrobial lipopeptid- carboxymethyl chitosan (BNAP-CMCS) nanoparticles added at different times on biofilm formation capability and the numbers of free bacteria and attached bacteria. Lastly, we tested the scavenging effect of BNAP-CMCS nanoparticles on biofilm formation and number of attached bacteria. The results showed that the amount of attached bacteria quickly increased over time and reached the maximum after 24 h of culture. The BNAP-CMCS nanoparticles had the greatest effect on biofilm inhibition at the concentration of 1 MIC, after 8 h of culture, and the effect was dose-dependent. The BNAP-CMCS nanoparticles had decreased also the numbers of free and attached bacteria in a dose-dependent fashion, after 8 hours of culture. The scavenging effect of BNAP-CMCS nanoparticles on free and attached bacteria was maximum at 6 MIC. In conclusion, lipopeptide carboxymethyl chitosan nanoparticles had a good inhibition and scavenging effect on the formation of Staphylococcus aureus biofilm and the growth of surface-attached bacteria.