This study was performed in order to isolate lactobacilli from chicken droppings and to select strains with the most promising probiotic properties. Lactobacillus strains were isolated from a flock of healthy laying hens. The first selection criterion was the ability to inhibit the growth of Salmonella Enteritidis. Then the tolerance to low pH and bile salt, the ability to coaggregate with pathogenic bacteria and hydrogen peroxide production were evaluated. Four isolates showing the best antagonistic activity against Salmonella Enetritidis were selected for further research. All isolates tested tolerated low pH and bile salt, likewise all produced hydrogen peroxide. They efficiently coaggregated with C. perfringens and relatively less with E. coli. Isolate 03’04 displayed above-average results in all criteria, thus it is considered as a potential probiotic for chickens, and will be further evaluated for health promoting effect in animals. The results presented in this study confirm the strain specific probiotic properties and prove the probiotic potential of isolate 03’04. Strong antagonistic properties against C. perfringens exhibited by certain Lactobacillus strains indicate the possibility to use them as a component of probiotic supplement in necrotic enteritis of poultry.