Thirteen presumptive thermo-tolerant Lactobacilli isolates were isolated from the small intestines of domestic chickens (Gallus domessticus) and were tested against various commercial antibiotics used in chicken feed to test antibiotic resistance of Lactobacillus spp. Isolates were identified by generating dendogram based on biochemical profiling results which was confirmed using PCR reaction. Bacteriocin produced by Lactobacillus sp. was optimized at different physicochemical factors which showed satisfactory zone of inhibition against pathogenic test organisms, both with and without purification. Four out of thirteen confirmed Lactobacillus sp. isolates showed resistance against Ciprofloxacin and one isolate showed resistance against erythromycin. A dendrogram was generated using biotyping where the isolates exhibited similarity to different species situated at same clusters and PCR assay confirmed isolates as Lactobacillus sp. upto genus level. Lactobacillus population will not decrease even after consumption of Erythromycin and Ciprofloxacin by humans to control gastroenteritis caused by enterobacteriace and probiotic activity of Lactobacillus sp. will not decrease and interfered by resistant antibiotics. Bacteriocin showed active bactericidal activity against E. coli, S. aureus, S. typhi, B. subtillis, B. megaterium, B. cereus, K. pneumoniae and E. fecalis. Future prospect involves purification of bacteriocin, MIC and MBC of produced Bacteriocin against test isolates.