The increasing resistance of microorganisms to antibiotics necessitates the search for new therapeutic strategies, including the use of nanoparticles with bioactive properties. The aim of this study was to investigate the antibiofilm and antimicrobial properties of metal and non-metal (Ag, Cu, Se) nanoparticles, obtained in deep eutectic solvent (DES), against resistant bacterial strains. The study investigated the determination of the minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) of selected Me-DES systems. The results showed that the MBCs of S. aureus ATCC 700699 and E. coli ATCC BAA -2523 for Ag-DES were 18.2 and 15.6 μg/mL, respectively, while those for Cu-DES and Se-DES were 125 μg/mL. Serial passages of bacterial cells were performed to assess the ability of microorganisms to develop resistance to the test systems, indicating their limited adaptability against Ag-DES and Cu-DES (MIC maintenance for 24 passages). The antibiofilm activity of the nanoparticles was confirmed by their ability to remove 12–37 % of S.Aureus bacterial biofilm and 23–51 % of E.coli biofilm after 24 h of exposure. In addition, the MTT cytotoxicity test showed that Me-DES systems exhibited low cytotoxicity against cell lines (A549). Analysis of the mobility of microorganisms after exposure to nanoparticles indicated a reduction in mobility capacity in the range of 14–68 % already at a concentration of 31.3 μg/mL, depending on the nanoparticles used. The study demonstrates that metal and non-metal nanoparticles in DES represent a promising system to combat resistant microorganisms, while reducing their ability to develop resistance and destroying the bacterial biofilm.