Steviol glycosides extracted from stevia rebaudiana plant are increasingly used as sweeteners. The minor steviol glycosides in the stevia extract, such as rebaudioside D and rebaudioside M, have superior sensory quality compared to the major components (e.g. rebaudioside A and stevioside). Due to the limited availability of rebaudiosides M and D in stevia leaf, they can be commercially produced by using enzymatic bioconversion and fermentation processes. The effect of steviol glycosides produced by different processes and their common metabolite steviol on human colonic bacteria is not adequately studied or published. There are limited research findings of the effect of artificial sweeteners, such as sucralose and Ace-K, on microbiomes. The key findings on artificial sweeteners are often extended, albeit incorrectly, to present the effect of all sweeteners including stevia, a natural sweetener on human microbiome. This study was designed to examine the potential influence of steviol glycosides and metabolite on the diversity and relative abundance of colonic bacteria genera in human fecal homogenates collected from adult male, female and young children. During the in vitro biotransformation of steviol glycosides to steviol as a final common metabolite in human fecal homogenate, the metabolic fate of steviol glycosides and the effect of stevia metabolites on the bacterial population were investigated by employing the next-generation genomic sequencing and informatics to record the bacterial diversity and relative abundance in the fecal microbiota. From in vitro anaerobic incubation samples, 20 major bacteria genera were observed, accounting for a range of 75% to 85% of the freshly collected colonic populations in male, female and children. Based on hierarchical clustering analysis, beta diversity differences were noted in bacterial genera between adult male, female and children fecal homogenate incubation samples as anticipated to be influenced by dietary variables and age as previously recognized by other researchers. The relative abundance of colonic bacterial populations in the incubation samples were different from the corresponding raw stool samples due to the influence of enrichment medium during fecal homogenate metabolic incubation. However, the principal coordinate analysis of bacteria genus data indicated a general lack of differences in alpha and beta diversity between samples incubated for 72 hours in the presence and absence of steviol glycosides and steviol metabolite amongst fecal homogenates collected from adult male, female and children. The relative abundance data (%) of 30 bacteria genus showed some effect of experimental incubation time, but there was no apparent difference between the fecal homogenate samples incubated in the presence and absence of steviol glycosides and steviol. The adult male, female and children bacteria genera data from this study have contributed to a conclusion that steviol glycosides used at typical low concentrations as a non-nutritive sweetener did not result in observable alternations of the bacterial population.