<p><strong>Background: </strong>The health benefits of probiotics are well established and known to be strain-specific. However, the role of probiotics obtained from different origins and their efficacy largely remains unexplored. The aim of this study is to investigate the <i>in vitro</i> efficacy of probiotics from different origins.</p> <p><strong>Methods: </strong>Probiotic strains utilized in this study include <i>Lactobacillus acidophilus</i> DDS-1 (human origin), <i>Bifidobacterium animalis ssp. lactis</i> UABla-12 (human origin), <i>L. plantarum</i> UALp-05 (plant origin) and <i>Streptococcus thermophilus</i> UASt-09 (dairy origin). Screening assays such as <i>in vitro</i> digestion simulation, adhesion, cell viability and cytokine release were used to evaluate the probiotic potential.</p> <p><strong>Results: </strong>All strains showed good resistance in the digestion simulation process, especially DDS-1 and UALp-05, which survived up to a range of 10<sup>7</sup> to 10<sup>8</sup> CFU/mL from an initial concentration of 10<sup>9</sup> CFU/mL. Two human colonic mucus-secreting cells, HT-29 and LS174T, were used to assess the adhesion capacity, cytotoxicity/viability, and cytokine quantification. All strains exhibited good adhesion capacity. No significant cellular cytotoxicity or loss in cell viability was observed. DDS-1 and UALp-05 significantly upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory TNF-α cytokine production. All the strains were able to downregulate IL-8 cytokine levels.</p> <p><strong>Conclusion: </strong>Of the 4 strains tested, DDS-1 demonstrated superior survival rates, good adhesion capacity and strong immunomodulatory effect under different experimental conditions.</p>