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Simple Summary Salmonella spp. continues to be one of the most important foodborne bacterial pathogens. S. enterica serotype Enteritidis (SE) that emerged as an important human illness during the 1980s is currently one of the most common non-typhoidal Salmonella serotypes worldwide. Poultry and their products (eggs and meat) are considered as one of the most important source of SE infection in humans. Due to restrictions in the addition of antibiotics in the feed of animals intended for human consumption, alternatives to these antibiotics have been sought. Probiotics have shown to reduce infection in turkey poults. However, studies are lacking to show how these probiotics influence the intestinal microbiome as well as how this microbiome is related to a lower infection by Salmonella. In the present study the effect of a Lactobacillus spp.-based probiotic on SE colonization was evaluated in two separate experiments. In both trials, a significant reduction in the incidence and log10 cfu/g of SE were observed in poults treated with the probiotic when compared with control poults (p ≤ 0.05). Results showed that the application of this probiotic culture could reduce SE cecal colonization in day-of-hatch turkey poults, although further research is needed to elucidate the mechanism of this response. Abstract The effect of Lactobacillus spp.-based probiotic candidates on Salmonella enterica serovar Enteritidis (SE) colonization was evaluated in two separate experiments. In each experiment, sixty-one day-of-hatch female turkey poults were obtained from a local hatchery. In both experiments, poults were challenged via oral gavage with 104 cfu/poult of SE and randomly allocated to one of two groups (n = 30 poults): (1) the positive control group and (2) the probiotic treated group. Heated brooder batteries were used for housing each group separately and poults were allowed ad libitum access to water and unmedicated turkey starter feed. 1 h following the SE challenge, poults were treated with 106 cfu/poult of probiotic culture via oral gavage or phosphate-buffered saline (PBS) to control groups. A total of 24 h post-treatment, poults were euthanized and the ceca and cecal tonsils from twenty poults were collected aseptically for SE recovery. In both trials, a significant reduction in the incidence and log10 cfu/g of SE were observed in poults treated with the probiotic when compared with control poults (p ≤ 0.05). The results of the present study suggest that the administration of this lactic acid-producing bacteria (LAB)-based probiotic 1 h after an SE challenge can be useful in reducing the cecal colonization of this pathogen in neonatal poults.