Objective: The diversity of bacteria and fungi during the processing of black powder was analyzed by high-throughput sequencing technology. Methods: For each part of the workshop for the sampling, use the Nanodrop samples quality after PCR amplification, the data quality control, through the sequence splicing, filtering, and optimizing sequence, and then OTU clustering and comments were conducted. Results: The relative content of Curtobacterium in grain flushing and mixing powder was higher, and baking had little effect on bacteria. Irradiation had a noticeable effect on reducing bacteria, and brachybacter could be reduced by order of magnitude after irradiation. The analysis of fungal species at the genus level showed that most of the fungi in the raw materials were sensitive to the high-temperature environment, and Auricularia had the highest relative content in the black powder, accounting for 97.99%, which indicated that baking could effectively reduce the number of fungi. Conclusion: This research provides technical support for bacteria and fungus control in the process of grain milling, improving the quality and safety and reference for the production of milling.