In the present study, the antibacterial and antibiofilm effects of Thymol-based chitosan nanogels were investigated. After clinical screening of MDR strains from the hospital environment, the morphological characteristics of the synthesized nanoparticles were identified using SEM, DLS, XRD and FTIR and the efficiency of encapsulation, stability and drug release were evaluated. The expression of OmpA and PgaB biofilm genes were determined by q-Real-Time PCR and the antibacterial and cytotoxic properties of the nanoparticles were determined by well diffusion and MTT methods, respectively. Nanoparticles with a size of 82.71 nm, encapsulation efficiency of 76.54% and stability up to 60 days at 4 °C were prepared. The results of the biological study showed strong antibacterial properties of Thymol-based chitosan nanoparticles by reducing the expression of OmpA and PgaB biofilm genes at a significant level of P ≤ 0.05 and reducing antibiotic resistance compared to the free drug thymol and chitosan nanogels. Thymol-based chitosan nanogels at concentrations of 0.125 to 256μg/mL showed the lowest cytotoxicity against HEK-293 compared to chitosan and free Thymol nanogels. The results showed very strong antibacterial properties of Thymol-based chitosan nanogels against MDR strains such as Staphylococcus, Acinetobacter and Pseudomonas as the challenging bacteria of the century.