Salmonella is a serious problem for both animal production and public health worldwide. Contaminated poultry is the main vehicle of Salmonella and the most important serotype is Salmonella enterica serovar Enteritidis. In order to test the efficiency of bacteriophages to treat Salmonella enterica serovar Enteritidis infections in poultry, a cocktail of two phages, F1055S and F12013S, isolated from chicken litter was applied by aerosol spray on fertile eggs challenged with Salmonella Enteritidis. To the best of our knowledge, this is the first experiment in which phages were applied by spray on fertile eggs. Two groups of eggs were challenged with Salmonella (3×10(8) colony-forming units [CFU]/mL) and one of them was treated with the phage suspension (2×10(6) plaque-forming units [PFU]/mL). A third group was used as nonchallenged and nontreated control. The phage treatment of challenged Salmonella eggs reduced the disease symptoms in the chicks. The arthritis and pasting after 8 days in the challenged and treated group were similar to those normally occurring in chicks (nonchallenged and nontreated chick control group) (p=1.000 and p=0.828, respectively, for arthritis and pasting) and were significantly lower than the challenged but nontreated ones (p=0.017 and p=0.002 for arthritis and pasting, respectively). The phage-treated group did not lose weight, showing an average weight similar to that of the nonchallenged control group and higher than that of the challenged nontreated group. The results of this study suggest that the application of phages by aerosol spray during the transfer of the eggs from incubators to hatchers may be an effective and inexpensive approach for reducing the horizontal transfer of Salmonella in poultry.