SummaryConjugated linoleic acid (CLA) is a mixture of linoleic acid isomers which have been shown to provide beneficial health effects; particularly, the isomers cis‐9, trans‐11 (c9‐t11) and trans‐10, cis‐12 (t10‐c12) are those with proven biological activity. Some lactic acid bacteria (LAB) have demonstrated the ability to synthesize CLA in culture media or in fermented milk, however, few studies have considered the possibility that LAB can continue to produce CLA in the human gut. Therefore, the objective of this study was to screen lactobacilli strains isolated from artisanal Poro de Tabasco cheese for their ability to biosynthesize CLA under simulated gastrointestinal digestion (SGD) and to evaluate their survival and gut colonization capacity under these conditions. First of all, LAB strains were identified molecularly and their CLA‐biosynthesis capacity in culture media and under simulated gastrointestinal conditions were evaluated. Besides, bacterial survival to the gastrointestinal conditions and gut colonization capacity by their adhesion and autoaggregation properties were assessed. Sixteen LAB strains were phenotypically and genotypically identified and were classified within the Lactiplantibacillus pentosus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Companilactobacillus farciminis species. Nine strains were able to synthesize CLA in culture media supplemented with linoleic acid (7.19‐18.78 μg/mL; with a conversion rate of 0.43‐1.13 %) and seven of them achieved it under simulated gastrointestinal conditions (4.28‐47.67 μg/mL; with a conversion rate of 0.51‐5.75%), with an outstanding performance of Lactiplantibacillus pentosus LC12. In addition, the viable counts demonstrated that all the study strains achieved to survive under gastric conditions with a high population survival rate of 94.5%, except for LC19 (67%); however all the nine strains recovered their viability with a high capacity to colonize the gut. Therefore, they may be considered as potential probiotic CLA‐producing strains, mainly Lactiplantibacillus pentosus LC12 since it was able to produce the closest CLA concentration to Limosilactobacillus reuteri ATCC 55739 (positive control). Thus, these strains can be used as starter cultures for designing promising functional foods with beneficial claims associated to the CLA content in the food matrix or produced after consumed.