The role of gut bacteria in animal health has became increasingly important, especially, with limitations use of antibiotics in animal feed due to consumer pressure and legislation. This study showed the relationships between gut bacteria and bird performance as a result of affecting by dietary supplementation of prebiotic such as (mannan oligosaccharide and lignin). A lower count of total aerobic bacteria was shown in mannan and lignin groups comparing with control during all experimental periods. Fortunately, count of the probiotic bacteria (Bifidobacterium spp. and Lactobacillus spp.)  increased significantly (P ≤ 0.01) in lignin and mannan groups compared to the control group at all three periods age, while the most increases were at 9 weeks. Determination of the susceptibility of isolated probiotics to antibiotics exhibited that the most effective antibiotic against the tested bacteria was ciprofloxacin (CIP), which reached up to 32 mm of inhibition zone, followed by vancomycin (VA) reaching up to 22 mm and finally tetracyclin (TE) which reached up to 20 mm. The antibacterial activity of the crude supernatant fluids obtained from lactic acid bacteria (LAB) culutures was generally greater against the Gram positive than the Gram negative tested bacteria. Sequencing of the 16S rRNA gene technique was considered to be a rapid and powerful method for identifying the probiotic isolates of Bifidobacterium spp. and Lactobacillus spp. to the species and at sub species level as Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis and Lactobacillus acidophilus.