To evaluate the ability of HN001 and BB536 to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. Twenty healthy Italian volunteers, eight males and twelve females, participated in the study. Ten subjects took a sachet containing 4 × 10 colony-forming units (CFU) of BB536 and 10 CFU of HN001, 30 min before breakfast (pre-prandial administration), while ten subjects took a sachet of probiotic product 30 min after breakfast (post-prandial administration). The ability of HN001 and BB536 to colonize human gut microbiota was assessed by means of quantitative real-time PCR, while changes in gut microbiota composition were detected by using Ion Torrent Personal Genome Machine. Immediately after 1-mo of probiotic administration, BB536 and HN001 load was increased in the majority of subjects in both pre-prandial and post-prandial groups. This increase was found also 1 mo after the end of probiotic oral intake in both groups, if compared to samples collected before probiotic consumption. At phyla level a significant decrease in abundance was detected immediately after 1-mo of BB536 and HN001 oral intake. This reduction persisted up to 1 mo after the end of probiotic oral intake together with a significant decrease of abundance if compared to samples collected before probiotic administration. Whereas, at species level, a higher abundance of , and was observed, together with a reduction of , , and abundance. In addition, during follow-up period we observed a further reduction in and , together with a decrease in and abundance. Conversely, the abundance of was increased if compared to samples collected at the beginning of the experimental time course. BB536 and HN001 showed the ability to modulate the gut microbiota composition, leading to a significant reduction of potentially harmful bacteria and an increase of beneficial ones. Further studies are needed to better understand the specific mechanisms involved in gut microbiota modulation.