Microbial resistance to antibiotics has become a global threat that interferes with the interaction among humans, the environment, and microbes. Our study sought to detect antibiotic-resistant bacteria in light of the growing interest in using antibiotics in the poultry sector. We collected 24 poultry liter samples from 04 different upazilas of Khulna district (Batiaghata, Dumuria, Paikgachha and Koyra). Initially, 09 bacterial isolates were selected and among them, 77.78% bacteria were found gram positive. Subsequently, to characterize these bacteria, a toal of 10 biochemical tests (methyl red test, MacConkey agar test, indole test, catalase test, triple sugar iron test, mannitol salt agar test, oxidase test, Voges-Proskauer test, citrate test and nitrate test) were carried out in this experiment. Moreover, our study also isolated and amplified bacterial DNA for Sanger sequencing and finally disclosed 09 different antibiotic resistant bacterial species, namely Priestia aryabhattai, Bacillus cereus, Priestia megaterium, Lysinibacillus macrolides, Rossellomorea aquimaris, Mammaliicoccus sciuri, Bacillus wiedmannii, Escherichia coli and Citrobacter freundii. Further, disk diffusion assays were performed following CLSI guidelines and it unveiled the susceptibility and resistant properties of isolates against commonly used 08 antibiotics (penicillin 10U, tetracycline 20µg, nitrofuran 300µg, clindamycin 2µg, azithromycin 15µg, quinolones 5µg, tetracycline 30µg and penicillin 30µg) in poultry farms. All of the bacterial isolates were found resistant to at least one of the antibiotics except C. freundii (isolate 9). Most of the isolates (66.67%) were resistant to nitrofuran, whereas all of them were susceptible to penicillin. Lastly, this study also made an effort to understand the evolutionary relationships of the identified species through a phylogenetic tree. Thus, the findings of this study will help farmers and common people to better understand the risk of developing antibiotic-resistant bacteria as a result of excessive antibiotic usage.