Raw Pu-erh tea (RAPT) was employed for kombucha fermentation using tea fungus obtained from different regions of China. The metagenomic sequencing revealed that Gluconobacter and Komagataeibacter were dominant bacterial genera and Brettanomyces and Starmerella were dominant fungal genera identified in the three samples. For volatile profile analysis, a total of 95 volatile compounds were identified in the three RAPT kombucha samples and 12 of them were recognized as aroma active compounds due to their high OAVs (≥1). The significant differences of volatiles in three samples were observed by the principal components analysis (PCA), which demonstrated that the flavor and sensory profile of RAPT kombucha were greatly influenced by the tea fungus. In addition, the antioxidant assay revealed that tea fungus fermentation could significantly (p < 0.05) enhance the bioactivity of RAPT due to the greatly (p < 0.05) increased total polyphenol content (TPC). •Raw Pu-erh tea (RAPT) was employed as new substrate for kombucha production.•The microbial composition and volatiles composition of RAPT kombucha were evaluated.•Key aroma compounds of three kombucha were identified and further quantified.•Significant differences in aroma and bioactivity were observed among tested kombucha.•The relationship between phytochemicals and the antioxidant capacity was evaluated.