Bacteria isolated from different segments of the gastro-intestinal tract (GIT) of healthy free-range broilers were screened for probiotic properties. Six strains were selected and identified as Lactobacillus gallinarum, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus crispatus, Enterococcus faecalis and Bacillus amyloliquefaciens based on 16S rRNA, gyrB and recA gene sequence analyses. All six strains produced exopolysaccharides (EPS) and formed biofilms under conditions simulating the broiler GIT. Lactobacillus johnsonii DPN184 and L. salivarius DPN181 produced hydrogen peroxide, and L. crispatus DPN167 and E. faecalis DPN94 produced bile salt hydrolase (BSH) and phytase. Bacillus amyloliquefaciens DPN123 produced phytase, amylase, surfactin and iturin A1. No abnormalities were observed when broilers were fed the multi-strain combination, suggesting that it could be used as a probiotic.