Lactobacilli are the most important bacteria gaining more consideration in the area of probiotics because they are generally regarded as safe. The present study was conducted to isolate and characterize Lactobacilli from avian microbiota and fermented milk products with the objective to evaluate these isolates as probiotics in broiler chicken. Various Lactobacillus species were isolated and, after characterization and screening, these isolated Lactobacilli were evaluated as probiotics in broiler chicken. A total 240 broiler chicks (day-old) were divided into 8 groups of 30 chicks each with 5 replicates. Six groups were administered isolated Lactobacilli in three dose rates and two routes. In addition, one group was administered commercial probiotics and another one group was maintained as control. Evaluation was done for the determination of effect on different performance parameters and immune response under experimental conditions. The chicks of experimental group, which was given Lactobacilli at dose rate 1x108 CFU/ kg body weight through drinking water, showed significantly (P<0.05) higher body weight, less feed consumption, decreased feed conversion ratio, low mortality and higher dressing percentage. This group also gained significantly (P<0.05) higher post-vaccinal antibody titres. Significantly, higher counts of Lactobacilli and lower count of E. coli were recorded in the intestinal contents of this group than other treated and control group. The treatment also showed similar results under field conditions. The study showed that these locally isolated Lactobacillus species had better effect as probiotics and these are as good as imported ones. It is recommended that these locally isolated Lactobacilli may be used as probiotics after species/strain identification through molecular methods and extensive field trials.