Development of the early gut microbiota is increasingly being recognised as an important factor for long-term overall health. Maternal transfer is an important source of bacteria for the neonatal intestinal microflora. Commercial poultry chicks, however, are hatched in a clean environment and have limited contact with parental microbiota. Bacterial seeding of the developing chick gut likely comes from feed and the environment, which may impact the development of a healthy gut microbiota profile. The present study investigated the capacity of five probiotic strains (Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus saerimneri) to colonise the developing chick intestine. At hatch, chicks were administered a single dose of bacteria orally. One week post administration cloacal swabs were collected and 77.7% were positive for L. reuteri, 72.2% for L. salivarius, 72.2% for L. crispatus, 88.8% for L. johnsonii, and 94.4% for L. saerimneri. Two weeks post administration, bacterial colonisation of the intestine was evaluated. All Lactobacillus strains successfully colonized the duodenum, jejunum, ileum, and caecum. Lactobacillus salivarius exhibited the lowest levels of colonization. Colonisation was evaluated again at 4 weeks of age. All Lactobacillus strains were detected in all segments of the intestine. Lactobacillus salivarius and Lactobacillus reuteri exhibited significantly lower loads in all intestinal segments compared with all other Lactobacillus strains. Lactobacillus johnsonii and Lactobacillus saerimneri exhibited the highest bacterial loads in all intestinal segments. An everted sleeve experiment was conducted using caeca from chicks to evaluate the effect of probiotic administration on Salmonella invasion into the epithelia. Caeca were everted on a sterile wooden swab and the internal contents were collected and combined with cell culture media. Salmonella Typhimurium was added and allowed to invade. Chicks administered a Lactobacillus strain exhibited significantly reduced Salmonella Typhimurium loads compared with control birds. Combined, these results demonstrate that the administration of Lactobacillus at hatch has the potential to shape the developing chick intestinal microbiota and contribute to reducing Salmonella Typhimurium colonisation.