Fructooligosaccharides (FOS) are one of the most studied prebiotics, selectively stimulating the growth of health-promoting bacteria in the host. However, there is increasing evidence that commensal gut bacteria, such as Bacteroides fragilis, Clostridium butyricum, Enterobacter cloacae, and even the pathogenic Escherichia coli BEN2908, are also able to metabolize FOS in vitro, and in some cases, FOS displayed adverse effects. Therefore, it is necessary to identify FOS-metabolizing species that are present in the gut. Unlike previous studies focusing on individual strains, this study used the traditional culture method combined with an alignment search on the gut bacteria database established from the Human Microbiome Project (HMP). The alignment results showed that homologous proteins for FOS transporters and glycosidases were distributed in 237 of the 453 strains of gut bacteria. La506 msmK encoding the ATP-binding protein and Aec45 fosGH1 encoding glycoside hydrolase were most widely distributed, in 155 and 55 strains, respectively. Seven of eight strains with both transporters and glycosidases were proven to be capable of metabolizing FOS, while five strains without either transporters or glycosidases were not. Fifteen species isolated from human feces and 11 species from the alignment search were identified to be FOS-metabolizing, of which Cronobacter sakazakii, Marvinbryantia formatexigens, Ruminococcus gnavus, and Weissella paramesenteroides are reported here for the first time. Thus, alignment search combined with the culture method is an effective method for obtaining a global view of the FOS-metabolizing bacteria in the gut and will be helpful in further investigating the relationship between FOS and human gut bacteria.