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Abstract Background The emergence of different viral infections calls for the development of new, effective, and safe antiviral drugs. Glycyrrhiza glabra is a well-known herbal remedy possessing antiviral properties. Objective The objective of our research was to evaluate the effectiveness of a newly developed combination of the probiotics Lactobacillus acidophilus and G. glabra root extract against two viral models, namely the DNA virus Herpes simplex virus-1 (HSV-1) and the RNA virus Vesicular Stomatitis Virus (VSV), with regards to their antiviral properties. Methodology To examine the antiviral impacts of various treatments, we employed the MTT assay and real-time PCR methodology. Results The findings of our study indicate that the co-administration of L. acidophilus and G. glabra resulted in a significant improvement in the survival rate of Vero cells, while also leading to a reduction in the titers of Herpes Simplex Virus Type 1 (HSV-1) and Vesicular Stomatitis Virus (VSV) in comparison to cells that were not treated. Additionally, an investigation was conducted on glycyrrhizin, the primary constituent of G. glabra extract, utilizing molecular docking techniques. The results indicated that glycyrrhizin exhibited a greater binding energy score for HSV-1 polymerase (− 22.45 kcal/mol) and VSV nucleocapsid (− 19.77 kcal/mol) in comparison to the cocrystallized ligand (− 13.31 and − 11.44 kcal/mol, respectively). Conclusions The combination of L. acidophilus and G. glabra extract can be used to develop a new, natural antiviral agent that is safe and effective.