The aim of this study was to characterize the probiotic characteristics of Lactobacillus fermentum strains isolated from Tulum cheese. Seven L fermentum strains were selected among the isolated and identified lactobacillus strains due to their abundance. When the gastric condition was considered, L. fermentum LP3 and LP4 were able to tolerate pH 2.5 and 1% bile salt. All L. fermentum strains had similar enzymatic activity and antibiotic resistance pattern but the highest antagonistic effect was detected within LP3, LP4 and LP6. Cholesterol assimilation amount of L. fermentum strains ranged between 12.1 and 45.3% in MRS and 20.7-71.1% in MRS with bile. The highest cholesterol assimilation in MRS and MRS with bile was occurred by LP3 and LP4, respectively. L fermentum LP2 adhered to caco-2 cells more than Lactobacillus rhamnosus LGG where LP3, LP4 and LP5 adhered at similar level. In conclusion, L fermentum LP3 and LP4 fulfilled sufficient criteria to be probiotics for use as a starter culture in the production of tulum cheese or other dairy products. Also this study indicated that some food-associated Lactobacillus strains non-predominant for gut biota have significant probiotic potential. (C) 2014 Elsevier Ltd. All rights reserved.