Background <p>Antimicrobial resistance (AMR) and its associated complications represent a major global health threat. Probiotics, among the limited available preventive strategies, may play an important role in reducing the risk of AMR.</p> Methods <p>A systematic review of studies assessing faecal antibiotic resistome in neonates who did versus did not receive probiotic supplementation. Databases were searched in October 2025.</p> Results <p>Eighteen studies (<i>n</i> = 3496) were included, comprising eight randomized controlled trials (RCTs) and ten observational studies (non-RCTs). Ten of the eighteen studies (RCTs: 5, non-RCTs: 5) reported significant reduction in the prevalence of faecal antibiotic resistome among probiotic supplemented infants. Five of the eight studies that reported no reduction relied on culture or polymerase-chain reaction-based methods rather than metagenomic analyses. No consistent associations were observed between probiotic dose and strain, or type of milk feeding and resistome colonization. Most included studies were assessed as having a low risk of bias. The certainty of evidence was rated as low to very low.</p> Conclusion <p>Probiotic supplementation may reduce faecal AMR gene colonization in neonates. Future RCTs should employ standardized study designs and include quantitative assessment of AMR gene abundance, along with clinically relevant outcomes such as sepsis and its associated complications.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>The first comprehensive systematic review focused on the effect of probiotics on neonatal fecal resistome and mobile genetic elements, incorporating evidence from 18 studies involving 3496 neonates.</p> </ItemContent> <ItemContent> <p>Suggests a potential role for targeted probiotic strategies as an intervention for reducing early neonatal antimicrobial resistance colonization, particularly in preterm infants at high risk of multi-drug resistance sepsis.</p> </ItemContent> <ItemContent> <p>Positions microbiome modulation as a strategy complementary to antibiotic stewardship in tackling global neonatal antimicrobial resistance.</p> </ItemContent> </UnorderedList></p>