Background and Objectives: Probiotics are added into the food or feed systems and provide beneficial effects to the human or animal health. This study aimed to isolate the gastrointestinal native Lactobacillus strains with high probiotic, cholesterol-assimilation and aflatoxin-degradation capabilities from native chickens. Materials and Methods: About 70 Lactobacillus isolates were isolated from ileum of the Fars province native chickens and were investigated for their probiotic properties. Results: Of 70 Lactobacillus isolates, 10 showed high probiotic capabilities, including survival at acidic conditions (pH up to 2.5), tolerance of 0.5% bile and 6-10% NaCl salts, growth in a wide range of temperature from 15 to 45°C, antagonistic effects against different important bacterial pathogens (Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, Clostridium defficile, Enterococcus hirae, Salmonella enterica and Staphylococcus aureus) and sensitivity to some important antibiotics. The selected strains had an aggregation time less than 120 min. The 16S-rDNA sequencing showed that the selected strains were highly related to Lactobacillus reuteri and L. casei. Finally, the selected strains in this study along with 10 other probiotic strains isolated and characterized in our pervious study were used to evaluate their cholesterol assimilation and aflatoxin B1 degradation capabilities. The potentials of cholesterol assimilation of the selected strains were significantly different (P<0.05) and ranged from 2.3% to 99%. The highest content of cholesterol assimilation was obtained in isolates M20 and M4 with more than 98% absorption. Moreover, four strains 43, OR7, M21 and OR9 were able to absorb AFB1 with 58.6%, 52.33%, 47% and 31.6% efficiency respectively. Conclusion: It could be concluded that the strains 43, M21 and OR7 showed high probiotic potentials for application in the poultry industry. [ABSTRACT FROM AUTHOR]