This study was to evaluate the potential of human intestinal bacterial species in the biotransformation of rutin to quercetin and isoquercitrin which is rarer than rutin in nature and could more potently inhibit the growth of some cancer cell lines. Bacterial strains isolated from healthy human fecal samples were identified through 16S rDNA gene sequence analysis and genome analysis. Isoquercitrin and quercetin were identified and quantified by UHPLC-QQQ-MS in multiple reaction monitoring mode. As results, the intestinal bacterial strains, comprising nine Gram-positive rods and one Gram-negative rod, were classified into Enterococcus, Lactococcus, and Escherichia genera. Among the ten isolates, Lactococcus garvieae Y3-2 and Lactococcus petauri Y5-4 produced higher amounts of quercetin compared to other bacteria. Interestingly, all strains of Enterococcus faecium species (Y4-1, Y4-2, Y5-1, and Y5-2) exhibited a relatively strong ability to convert rutin to isoquercitrin, with Y4-2 being particularly efficient. The higher L-rhamnosidase activity observed in E. faecium Y4-1 and E. faecium Y4-2 correlated with their significant yield of isoquercitrin. Four or three genes were probably involved in rutin metabolism according to the analysis of flavonoid pathway based on genome sequences. The results provided information for selecting bacterial species to convert rutin into target bioactive compounds, and for purification of pure enzymes to biosynthesize isoquercitrin.