Mounting evidence suggests that distinct microbial communities reside in tumors and play important roles in tumor physiology. Recently, Nejman et al. profiled the composition and localization of intratumoral bacteria using 16S DNA sequencing and histological visualization methods across seven tumor types, including human glioblastoma. However, considering potential contamination in their sample origins and processing, the results based on traditional histological methods need to be validated. Here, we propose a three-dimensional (3D) intratumoral microbiota visualization and quantification protocol to observe microbiota in intact tumor tissues on the premise of avoiding possible contamination in the surface of tissues, based on tissue clearing, immunofluorescent labeling, microscopy imaging, and image processing. For the first time, we have achieved 3D quantitative imaging of bacterial LPS fluorescent signals deep in gliomas in a contamination-free manner, which was founded mostly localized near nuclear membranes or in the intercellular space. Through an automated statistical algorithm, reliable signals can be distinguished for further analysis of their sizes, distribution, and fluorescence intensities. Combining two-dimensional images from multiple thin-section histological methods, including immunochemistry and fluorescence in situ hybridization, we provide a comprehensive histological investigation of the morphology and distribution of these signals on human glioma samples. We expect that this multi-evidence chain will provide supporting proof for the presence of intratumoral bacteria in human glioma and that the integrated pipeline can be applied to investigate the native bacteria within diverse tumors and contribute to the interpretation of their direct roles in the tumor microenvironment.