The present study was conducted to isolate, identify and characterize the bacterial pathogens present in feces of tiger and lion of Dhaka Zoo and their antimicrobial activities during the period from July to December 2013. 42 (twice from 6 tiger and 15 lion) faecal samples were collected from the cages of animals just after defecation and brought to the Microbiology laboratory of the Department of Microbiology, Hajee Mohammad Danesh Science and Technology Universiy, Dinajpur for bacteriological examination. Isolation and identification of the microorganisms were performed by their morphology on different cultural media, staining characteristics and biochemical tests. Then, the isolated bacteria were characterized by antimicrobial susceptibility analyzed by the online software ABIS Encyclopedis/tgw1916, 2014 and detected by polymerase chain reaction (PCR) for molecular detection of E. coli, Salmonella and Staphylococcus sp. Out of 42 samples 17 (40.47%) samples were positive for E. coli, 15 (35.71%) samples were positive for Salmonella sp, 18 (42.85%) samples were positive for Staphylococcus sp. The antibiogram study revealed that most of the E. coli, Salmonella spp. and Staphylococcus spp. were resistant to Doxycycline, Norfloxacin, Ciprofloxacin, Neomycin, Enrofloxacin, Ciprofloxacin, Amoxicillin, Levofloxacin and Tetracycline. However, most of the E. coli, Salmonella sp. and Staphylococcus sp. were susceptible to Neomycin, Gentamicin and Colistine sulphate which indicate that the use of these antimicrobial may be chosen in clinical control of Salmonella, E. coli, and Staphylococcus sp infection. PCR results confirmed 7 (3 E. coli and 4 Salmonella) isolates out of 11 in tiger and lion. Most of the isolates have zoonotic importance and confirmed the presence of either commensal or pathogenic for tiger and lion in zoo.