Nowadays, most of the traditional and conventional antibiotics are not effective against drug resistant bacterial strains. The emergence and spread of drug resistant bacterial cells calls for new therapeutic agents and strategies to control and treat the infections caused by these bacteria. In this regard, the application of nano-technology in medicine is an interesting approach. Therefore, the aim of this study was to evaluate the antibacterial and anti-biofilm activities of Ag Np conjugated to chitosan against multidrug resistant isolates of Staphylococcus aureus and Acinetobacter baumannii. Synthesis of the Ag Np and chitosan Np were performed according to the Turkevich and Ionic gelation methods, respectively. Then conjugation of nanoparticles was carried out by standard method. FTR analysis and transmission electron microscopy were used for validation of nanoparticle conjugation. Twenty clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and 20 clinical isolates of carbapenem resistant Acinetobacter baumannii (CRAB) were obtained from the microbial bank of Imam Khomeini hospital of Tehran, Iran. MIC values of the nanoparticles alone and in conjugation were determined using the microbroth dilution method. Then, fractional inhibitory concentration of agents was concluded by standard method. Finally, anti-biofilm activities of the conjugated Ag Np-chitosan at sub-MIC concentrations was tested against bacterial isolates. Synthesis of Ag Np resulted in the formation of nanoparticles with 10 nm dimensions. MIC90 of the chitosan, Ag Np and their conjugated form were respectively 64, 16, and 8 μg/mL against CRAB isolates. Also, MIC90 of the tested agents, in the same order as mentioned above, were 32, 16, and 4 μg/mL against MRSA. Combination of the agents had additive (0.625) and synergistic (0.375) effects against CRAB and MRSA. Ability of biofilm formation was dramatically reduced by ¼ MIC concentration (2 μg/mL) against the CRAB and ½ MIC concentration (2 μg/mL) in the case of the MRSA isolates. Ag Np-chitosan conjugation, an ideal alternative for ineffective antibiotics, exhibits great antibacterial and anti-biofilm effects against CRAB and MRSA isolates.