Xiaguan Tuo Tea is largely consumed by the Chinese, but there is little research into the microbial diversity and component changes during the fermentation of this tea. In this study, we first used fluorescence in situ hybridization (FISH), next-generation sequencing (NGS) and chemical analysis methods to determine the microbial abundance and diversity and the chemical composition during fermentation. The FISH results showed that the total number of microorganisms ranges from 2.3×102 to 4.0×108 cells per gram of sample during fermentation and is mainly dominated by fungi. In the early fermentation stages, molds are dominant (0.6×102~2.8×106 cells/g, 0~35 d). However, in the late stages of fermentation, yeasts are dominant (3.6×104~9.6×106 cells/g, 35~56 d). The bacteria have little effect during the fermentation of tea (102~103 cells/g, <1% of fungus values). Of these fungi, A. niger (Aspergillus niger) and B. adeninivorans (Blastobotrys adeninivorans) are identified as the two most common strains, based on Next-generation Sequencing (NGS) analysis. Peak diversity in tea was observed at day 35 of fermentation (Shannon-Weaver index: 1.195857), and lower diversity was observed on days 6 and 56 of fermentation (Shannon-Weaver index 0.860589 and 1.119106, respectively). During the microbial fermentation, compared to the unfermented tea, the tea polyphenol content decreased by 54%, and the caffeine content increased by 59%. Theanine and free amino acid contents were reduced during fermentation by 81.1 and 92.85%, respectively.