The present research was accomplished to characterize probiotics from broiler gastrointestinal tract (GIT) by profiling biochemical, antimicrobial, and antibiotic sensitivity properties. Eventually, probiotic potentiality was evaluated as a substitute for antibiotic supplements in broiler focusing growth performance, carcass characteristics, and serum lipid profile. Probiotic bacteria were characterized based on morphological, physiological, and several biochemical tests. Antibacterial activity against a broad spectrum of antibiotics and bacterial pathogens was detected. An in vivo trial was conducted on 40-day-old Ross 308 broiler strains during 21 days in an in vivo trial. The chicks were divided into total of five groups, a control group and four experimental groups (Antibiotic1, Antibiotic2, Probiotic1, and Probiotic2) in a completely randomized design. Probiotic was supplemented in broiler feed (2× 109 CFU/g feed) or by direct oral gavage (1× 109 CFU/chick). The variables of production performance like body weight (BW), average daily gain (ADG), feed intake (FI), and feed conversion ratio (FCR), carcass characteristics and serum lipid profile were measured. 10 probiotic bacteria were presumptively identified as Lactobacillus sp. based on the morphological, physiological, and strong resistance properties in several biochemical tests. The mixture of Lactobacillus had favorable effects on productive performance of broilers regarding BW, ADG, and FCR (p < .05) compared with chickens that had no additive or had antibiotic during overall period of in vivo trial. Additionally, noteworthy efficacy on carcass characteristics and serum lipid profile were found (p < .05) in Lactobacillus mixture fed chicken groups of in vivo trial. Mixed Lactobacillus sp. can be considered as a potential additive for broiler diet attributable to noteworthy efficacy on growth performance, carcass characteristics, and serum lipid profile. Accordingly, the research highlights the need for suitable alteration of antibiotics through probiotic characterization and proper inclusion in broiler diet.