Bacterial inoculation stimulates growth and adaptation in micropropagated plants. This study evaluated the effects of biotization on in vitro seedling production and in vivo adaptation in two potato cultivars, Agatha and Duvira. Nine bacterial strains were tested for hormone production and ACC deaminase activity and then inoculated in vitro and re-inoculated in vivo. Growth, adaptation, and tuber production were assessed. Biotization significantly enhanced seedling growth, survival, and tuber yield. Biotized seedlings had a 1.3-fold higher survival rate than the controls. Azospirillum brasilense Ab-V5 and Rhizobium tropici CIAT 899 promoted at least one growth variable in both cultivars under in vitro and in vivo conditions. A. brasilense Ab-V5 consistently improved plant performance across production stages, with re-inoculated plants showing 1.2–1.3-fold increases in stem and root length and a 1.1-fold gain in total dry biomass. Additionally, inoculated plants produced 1.9 times more tubers than the controls. Biotization effects were strain-dependent, with A. brasilense Ab-V5 improving in vitro seedling quality and enhancing plant performance and survivability in vivo.