Steady advances in our knowledge of the composition and function of the human microbiome at multiple body sites including the gut, skin and airways will likely contribute to our understanding of mechanisms of probiotic action by beneficial microbes. Microbe:microbe and microbe:human interactions are important considerations as we select probiotics for pediatric patients in the future. Although our knowledge about the composition of the microbiome is progressing rapidly, many gaps exist about the functional capacity and metabolic machinery of the human microbiome. Based on a limited amount of data, probiotics appear capable of altering the composition and function of the microbiome. Probiotics may be part of dietary strategies that combine ways to enhance microbiome function with nutrients that may be converted to active compounds promoting human health. Probiotics have yielded beneficial effects in numerous studies in the context of different diseases in pediatric gastroenterology. These disease states include necrotizing enterocolitis, antibiotic-associated diarrhea and colitis, acute gastroenteritis and irritable bowel syndrome. In the skin and airways, it is unclear if probiotics can affect the function of the microbiome to reduce the impact of diseases such as asthma and atopic dermatitis. An enhanced understanding of the effects of probiotics on the microbiome should facilitate selection of optimal probiotic strains for specific diseases in the future.