Bifidobacterium animalis subsp. lactis bb12 and B. longum 20099 were used to hydrolyze isoflavone glycosides (IG) to biologically active forms, which are isoflavone aglycones (IA), in soymilk (SM) prepared from soy protein isolate (SPI) and SM supplemented with 0.5% (w/v) of lactulose (SML). Supplementation of lactulose significantly ( p < 0.05) enhanced the viable counts of B. animalis subsp. lactis bb12 and B. longum 20099 up to 2.34 and 2.15 log CFU/mL, respectively. Bifidobacterium animalis subsp. lactis bb12 and B. longum 20099 utilized 3.32 and 3.75 mg/mL of lactulose at 24 h of incubation, respectively. Supplementation of lactulose also appeared to be a key factor in decreasing the pH of SML. The biotransformation of IG to IA was enhanced significantly by 6.8-17.1% and 12.8-13.5% in SML by B. animalis subsp. lactis bb12 and B. longum 20099, respectively. However, the presence of lactulose in SML showed the stimulating effect on B. longum 20099 only after 12 h of incubation. Isoflavone aglycones ranged from 69.5 to 77.1% of total isoflavone compounds in SML after incubation.