The aim of vaccinating layer flocks is to reduce the shedding of wild-type Salmonella in poultry environment, thus reducing the incidence of human salmonellosis. In the current study, a commercially available live attenuated vaccine (Vaxsafe ST) was reconstituted in buffered peptone water, Marek’s diluent and water and three different doses (107, 108 and 109 CFU/chick) were assessed for their effects on gut microbiota and gut colonisation potential in layer chicks. The vaccine diluent and dosage minimally affected alpha diversity and overall there was no significant difference between different diluents. Although heavily overlapped between the treatment groups beta diversity was significantly different based on the vaccine diluent and dose, suggesting that the vaccinated chickens microbial community was different from unvaccinated ones. The vaccine dosage and diluent affected microbial genera in abundance included Blautia, Colidextribacter, Dickeya, Enterococcus, Eubacterium_hallii_group, Lactobacillus, Pediococcus and Sellimonas. The abundance of Colidextribacter was significantly lower in the vaccinated groups that received vaccine in Marek’s and water diluents, while Lactobacillus abundance was significantly lower in the water group. Higher vaccine dose (109 CFU/chick) did not significantly reduce the abundance of microbial genera. Chick’s age significantly affected the abundance of microbial genera than vaccine dose and diluent. The abundance of Lactobacillus, Blautia, Caproiciproducens, Pediococcus and Colidextribacter was significantly higher on day 14 compared with day 7 of chicks age. The Vaxsafe ST load in the caeca was not significantly affected by diluent and vaccine dose; however, it was significantly lower at day 14 compared with day 7 of chick’s age. Overall, Vaxsafe ST vaccine had minimal effects on the gut microbiota diversity and composition in layer chicks, whereas age had significant effects on gut microbiota.