The impact of five human milk oligosaccharides (HMOs)-2'-fucosyllactose (2FL), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT)-on the gut microbiota and short-chain fatty acid (SCFA) metabolites in infants aged 0-6 months was assessed through in vitro fermentation. Analyses of the influence of different HMOs on the composition and distribution of infant gut microbiota and on SCFA levels were conducted using 16S rRNA sequencing, quantitative real-time PCR (qPCR), and gas chromatography (GC), respectively. The findings indicated the crucial role of the initial microbiota composition in shaping fermentation outcomes. Fermentation maintained the dominant genera species in the intestine but influenced their abundance and distribution. Most of the 10 strains effectively utilized HMOs or their degradation products, particularly demonstrating proficiency in utilizing 2FL and sialylated HMOs compared to non-fucosylated neutral HMOs. Moreover, our study using -dominant strains and -dominant strains as inocula revealed varying acetic acid levels produced by upon HMO degradation. Specifically, the -dominant strain yielded notably higher acetic acid levels than the -dominant strain ( = 0.000), with minimal propionic and butyric acid production observed at fermentation's conclusion. These findings suggest the potential utilization of HMOs in developing microbiota-targeted foods for infants.