The species diversity, abundance, and location of intestinal bacteria bear major consequences on host intestinal functions, affecting the balance between health and disease. Recent studies addressed the relative abundance of bacteria in feces and on the gut mucosal surface. However, few of these publications reported the absolute number of bacteria in different compartments of the gut, especially those associated with the intestinal epithelial surface, compared to the luminal content reflected by feces. In this study, the number of bacteria present in the lumen or associated with the mucosal surface of mice gut samples was calculated using the bacterial “internal standard method” during sequencing. Three spiking bacteria were used for mutual corroboration. The microbiota spatial distribution in the cecum, proximal and distal colon were studied in the lumen content and gut wall fixed niches. This study also provides an example to demonstrate that absolute and relative quantification may yield different conclusions. This study’s method may eliminate such misjudgments and provide insight into the distribution and quantity of gut microbiome.