Newly hatched broiler chickens are the most susceptible to Salmonella infections, especially during the first 24h. At this age, the gut microbiome is not fully developed and offers little protection in the form of competitive exclusion. In this study, one group of newly hatched, Salmonella-free broilers were colonised with a multi-species probiotic (2.0 × 1010 to 8.9 × 1010CFU per kg feed) for 28days, consisting of Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus gallinarum, Lactobacillus johnsonii, Enterococcus faecalis and Bacillus amyloliquefaciens. Broilers in another group received oxytetracycline (200mg/kg feed), instead of the probiotic, for 28days. On days 9 and 10, broilers in both groups were gavaged with 9 × 107CFU Salmonella Enteritidis A9, a pathogenic strain isolated from infected broilers. On day 14, Salmonella was detected in the ceca of 95% of broilers treated with the multi-species probiotic, but 2weeks later, almost half of the birds (45%) had no Salmonella in their ceca. Similar results were recorded after 28days of treatment with oxytetracycline. Only 10% of Salmonella-infected birds not treated were Salmonella-free on day 28. Growth performance, immune organ weight (spleen and bursa of Fabricius) and whole blood cell counts of birds treated with the multi-species probiotic and oxytetracycline, respectively, were similar to untreated and uninfected birds throughout the 28-day trial (p > 0.05). On day 14, serum lysozyme levels of broilers exposed to the probiotic were lower (8.0μg/mL) compared with those of broilers treated with oxytetracycline (11.0μg/mL). Although the multi-species probiotic and oxytetracycline stimulated the immune system, probiotics are safer to use than antibiotics and should be the preferred choice of treatment.