Potato virus Y (PVY) is causing a serious loss in yield and quality of potatoes. The present study shows the effect of antiviral Ribavirin and antibacterial Bacteriocin on regeneration response and production of PVY-free plants under in vitro conditions. PVY-infected and healthy potato meristem tips were transplanted in Murashige, & Skoog, (MS) medium supplemented with 0.1 mg L−1 GA3, 0.1 mg L−1 NAA, and 500 mg L−1 malt extract, ribavirin or/and Bacteriocin and the acquired resistant and reduction of virus in plantlets were determined. DAS-ELISA and RT-PCR were used to index the mother plant and in vitro-regenerated plantlets for virus indexing.  In vitro, regenerated plantlets tested negative in both ELISA and RT-PCR were only considered as virus-free. The bacterial and fungal contaminants were isolated and identified based on morphological and cultural characteristics of major genera that included, Pseudomonas, Bacillus, Serratia, Xanthomonas, Aspergillus, Penicillium, Geotrichum, Trichoderma, and Alternaria. MS medium treated with ribavirin or Bacteriocin suppressed contaminated bacteria and fungus. Consequently, plant height, leaf size, number of branches, and root number all significantly increased, in addition to plantlet growth. In addition, PVY-free potato plantlets were produced, as validated by the DAS-ELISA analysis. For more, When compared to untreated plantlets, bacteriocin and/or generate inducers enhanced protein content and oxidative enzyme activities (PO, PPO), salicylic acid, Chl a, Chl b, and carotenoids in potato healthy plantlets and PVY-infected plantlets. The current study suggested that the use of ribavirin and/or Bacteriocin improves the quality of potato plantlets grown in tissue culture.