Bacteria are one of the major causes of severe infections and diseases of plants and animals. Salmonella are crucially important due to infection in poultry leading to huge economical loses. Due to high cost and microbial resistance to the currently available chemical antibiotics, demand of screening natural products with antibiotics effects is increased. Plants are rich sources of natural bioactive compoundswith antibiotic effects. Saponins are natural compounds of plant sources having a diverse range of applications. In present study we investigated the in vitro and in vivo antibacterial activities of green tea seed extracted saponins. Green tea seeds crude extract was prepared in 70% ethanol by continuous reflux in heating mantel for 5 hours. Crude saponins were extracted from the crude ethanolic extract of green tea seed by column chromatography using macroporous resin (D101). Saponin mixture in fraction 1 (Fr1) was obtained from crude saponins extract via column chromatography. Fr2 and Fr3 were isolated from saponins mixture by preparative HPLC. Antibacterial activities of the isolated saponins fractions were investigated against Escherichia coli (ATCC 25922), Streptococcus aureus (ATCC 12600), and six serovars of Salmonella. In vitro antibacterial activities were determined by disc-diffusion method and growth inhibition in liquid culture using 96-well plate. Results showed that the green tea isolated saponins fractions possess antibacterial effects in the following order Fr1> Fr2> Fr3. Antibacterial mechanism of saponins was elucidated by cell wall and membrane damaging potential of saponins determined by measuring AKP and soluble proteins levels. Fr1 was further used for in vivo antibacterial activities. Five-week grown chickens were selected for in vivo work, divided into three groups as control, infected, and treatment groups. Infected and treatment groups chickens were infected with bacteria and only treatment group chickens were treated with saponins. The qRT-PCR analysis of the blood and feces samples of the different groups' animals shows the presence of bacteria only in infected group while reduced expression levels of the bacterial pathogens were found in the samples of treatment group. Our results demonstrated that the green tea seed saponins used in this study possess strong antibacterial activities.