The gut microbiome is a key part of infant health, and can be influenced by feeding method. Breastmilk and commercial formula contain markedly different concentrations of sialylated human milk oligosaccharides (HMOs), which are solely digested by gut microbes via microbial sialidases. The free sialic acid is then accessible to local intestinal flora or reabsorbed by the infant for development. It is critical to understand the diversity in structure and function of microbial sialidases and apply this knowledge to early gut microbiome colonizaHon. We examined a large human fecal microbiome metagenomic database and identified microbial sialidases based on a protein structure-defined rubric. Then, a sequence similarity network containing 290 unique enzymes was created, from which nine diverse enzymes were selected for recombinant expression and purification. Enzyme activity was evaluated with the general reporter substrate 4MU-neuraminic acid and several biologically relevant sialylated HMOs. A wide range of selective substrate processing abilities were observed, and a moderate overall preference for 3’-linkages was noted. These activity data were complemented by the determination of novel x-ray crystal structures of sialidases from Alis<pes dispar and Parabacteroides merdae. Structural analyses pinpoint two variable loop regions near the active site that begin to explain key observed functional differences. Finally, anaerobic culturing of B. thetaiotaomicron with sialylated HMOs revealed that sialidases are necessary for microbial growth on these sugars. These findings provide critical insights into HMO utilization for early life colonization, and may guide the improvement of commercial formulas to more effectively imitate the compounds in human breast milk.