Probiotics are increasingly administered to premature infants to prevent necrotizing enterocolitis and neonatal sepsis. However, their effects on gut microbiome assembly and immunity are poorly understood. Using a randomized intervention trial in extremely premature infants, we tested the effects of a probiotic product containing four strains of Bifidobacterium species autochthonous to the infant gut and one Lacticaseibacillus strain on the compositional and functional trajectory of microbiome. Probiotic treatment accelerated the transition to a mature, term-like microbiome with higher stability and species interconnectivity. Besides infant age, probiotic Bifidobacterium strains and stool metabolites were the best predictors of microbiome maturation, and structural equation modeling confirmed probiotics as a major determinant for the trajectory of microbiome assembly. Probiotic-driven microbiome maturation was also linked to an anti-inflammatory intestinal immune milieu. This demonstrates that Bifidobacterium strains are ecosystem engineers that lead to an acceleration of microbiome maturation and potential immunological benefits in extremely premature infants.