This study sought to determine the fermentation potential of human milk oligosaccharides by mixed cultures of fecal microbiota from breast-fed (BF; n = 4) and formula-fed (FF; n = 4) infants. Infant fecal inocula were incubated with galactooligosaccharide (GOS), gum arabic (GA), HP inulin (HP), 2'-fucosyllactose (2'FL), 6'-sialyllactose (6'SL), and lacto-N-neotetraose (LNnt). GOS, 2'FL, and LNnT had a lower pH than other substrates after 3 h (P < 0.05). Total short chain fatty acids were greater in FF compared to BF infants at 6 h (P = 0.03) and 12 h (P = 0.01). GOS, 2'FL, and LNnT led to more lactate than 6'SL, HP, and GA (P < 0.05). Bifidobacteria populations were greater (P = 0.02) in FF at 6 and 12 h. Overall, GOS, 2'FL, and LNnT were rapidly fermented by infant fecal inocula, 6'SL and HP had intermediate fermentability, while GA had little fermentation. Inocula from FF infants fermented substrates more rapidly than inocula from BF infants, which should be accounted for when evaluating substrate fermentability. These data will aid in future infant formulas to promote optimal gut health in FF infants.