This study was conducted to investigate the antimicrobial-biofilm activity of chitosan (Ch-NPs), silver nanoparticles (Ag-NPs), ozonated olive oil (O -oil) either separately or combined together against endodontic pathogens. While testing the antimicrobial activity, Ch-NPs showed the least minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values exerting eightfold higher bactericidal activity than O -oil against both and as well as fourfold higher fungicidal activity against . Antimicrobial synergy test revealed synergism between O -oil and Ch-NPs against the test pathogens (FIC index ≤ 0.5). Ch-NPs was superior at inhibiting immature single and mixed-species biofilm formations by 97 and 94%, respectively. Both of O -oil and Ch-NPs had a complete anti-fibroblast adherent effect. The safety pattern results showed that O -oil was the safest compound, followed by Ch-NPs. The double combination of Ch-NPs and O -oil reduced the mature viable biofilm on premolars model by 6-log reductions, with a fast kill rate, indicating potential use in treating root canals. Therefore, the double combination has the potential to eradicate mature mixed-species biofilms and hence it is potent, novel and safe.