The objective of this study was to evaluate the probiotic properties of Lactobacillus strains isolated from mother's milk and their effects on cholesterol assimilation. In this study 120 isolates from mother's milk were phenotypically and genotypically characterized. Among these, only 6 predominant strains were identified as Lactobacillus spp. The following parameters were selected as important test variables in model stomach passage survival trials: acid and bile tolerance, antimicrobial activity, antibiotic susceptibility and cholesterol reduction. Results showed that the considerable variation existed among six strains. Moreover, the strain HMI68 is the most acid-tolerant and the HMI28 and HMI74 is the most acid-sensitive of all strains tested. HMI118 did not grow at 0.5% and 1% bile concentration after 5 h but the HMI68 and HMI43 showed some tolerance to such bile concentration. The differences found in the growth rate were not significant (P > 0.05). HMI68 showed resistance to most of the antibiotics as well as antagonistic activity against the tested pathogens. The amount of cholesterol reduction is increased when the media supplemented with bile salts. HMI68 assimilate 61.05 ± 0.05 μg/ml cholesterol with the presence of 0.3% bile salt this could be significantly decreased by 25.41 ± 1.09 μg/ml without bile salt. HMI68 was identified to be Lactobacillus oris HMI68 and 16S rRNA sequence was deposited in the National Center for Biotechnological Information (GenBank). For the first time the cholesterol-reducing property of L. oris isolated from mother's milk were investigated in this study. Therefore the effective L. oris HMI68 strain was regarded as a candidate probiotic.