Selenium, a trace element with antioxidant properties, plays a vital role in the metabolism of microorganisms. Meanwhile, supplementation with selenium may also modify the activity of probiotics. Thus, this study aimed to analyze the effect of selenium sup- plementation on the growth, tolerance, and selenium binding capacity of two probiotic strains: Lactiplantibacillus plantarum DSM24730 and 299v. In particular, this study aimed to determine whether introducing this microelement into the culture environment would affect the growth capacity and detoxification mechanisms of these bacteria. Additionally, the ability of the selected strains to absorb and store selenium was analyzed, which could have potential benefits for both human health and the quality of probiotic preparations. Methods: Bacterial cultures of Lactiplantibacillus plantarum DSM24730 and 299v were grown in specially prepared media supplemented with different concentrations of selenium (0–100 mg/L). This study assessed several key physiological parameters of microorganisms in real- time, including biomass production, growth dynamics, and the ability to survive in conditions that simulate the gastric and intestinal environments. Results: Growth curves and biomass analyses revealed that moderate selenium concentrations (5–10 mg/L) supported the growth of both strains, whereas higher concentrations (50–100 mg/L) inhibited biomass production and delayed the onset of growth, especially in DSM24730. Intensive growth of bacterial biomass (0.23 g/L; p < 0.05) in the experimental medium supplemented with 5 mg Se4+/L was observed for L. plantarum 299v after 24 h of cultivation. In the case of the DSM24730 strain, the lag phase (∆tlag) was prolonged at higher selenium concentrations, reaching 12 h at 100 mg/L, while the logarithmic phase (∆tlog) was shortened from 12 h in the control medium to only 2 h at 100 mg/L. The 299v strain demonstrated faster growth, higher biomass yield, and more rapid selenium uptake at moderate concentrations, while DSM24730 accumulated higher final levels of selenium after prolonged incubation. The highest selenium content (0.45 mg Se4+/g; p < 0.05) after 72 hours of cultivation was accumulated by strain L. plantarum 299v. Meanwhile, after the same culture time, the second bacterial strain (DSM24730) accumulated only 0.29 mg Se4+/g; p < 0.05). Tolerance assays using simulated gastric and intestinal fluids demonstrated that both strains survived under acidic gastric conditions; however, the viability of these strains significantly declined in intestinal juice at selenium concentrations of ≥10 mg/L, indicating an apparent dose- dependent inhibitory effect. Conclusions: These observations suggest that 299v is more efficient in rapid selenium assimilation and biomass formation, while DSM24730 may be more suitable for high-capacity selenium loading over time. These findings are consistent with other studies on selenium-enriched probiotics, highlighting strain-specific responses to selenium supplementation. Strain selection and selenium dose optimization are essential for developing safe and effective selenium-enriched probiotic products.