This study reports the probiotic attributes of Lactobacillus strains isolated from chicken faeces and mainly their capabilities to prevent infectious diseases and improve chicken production performance. Thus, 22 Lactobacillus strains were isolated from 50 chickens’ faeces samples and assessed for their resistance to gastric acidity (pH 0.5, 1, 1.5, 2 and 2.5), tolerance to bile salts, adherence to broiler intestinal cells and antibacterial activity. These in vitro screening analyses revealed Lactobacillus plantarum S22 and L. plantarum S27 as the only strains capable to survive at pH 2.0 in MRS broth (log 10 cfu/ml=5.02 and 8.46 log respectively), while the remaining strains were not resistant to pH≤2.0. Similarly, 21 strains were resistant to bile at 0.5% (log 10 cfu/ml=0.09-3.32 log), but only Lactobacillus fermentum S26, L. plantarum S22 and L. plantarum S27 were able to grow in the presence of 0.1% (w/v) bile (8.23±0.15; 8.39±0.17 and 8.57±0.07 respectively). Most of these isolates (19/22) were active against Escherichia coli ATCC 25922, E. coli SL2016 and Salmonella enterica CIP 81-3. Lactic acid is likely the main antibacterial compound produced since the neutralised supernatant was devoid of any antibacterial activity . In vitro characterisation of these 22 novel strains, based on the aforementioned criteria revealed L. plantarum S27 as the most suitable strain for in vivo analyses. To this end, this strain was assessed for its sensitivity to different antibiotics and adhesion to poultry intestinal cells to ascertain it probiotic attributes. The administration of L. plantarum S27 to the chicks at 10 9 cfu/ml permitted to improve the animal food intake and weight. Taken together, data from in vitro and in vivo analyses indicated that L. plantarum S27 might be a worthy probiotic for chickens rather than adding antibiotics to animals feeding.