In this study, chemical analysis and membership function were employed to systematically compare the physicochemical properties of Nongxiangxing Daqu of different grades (premium, first and second) at the middle stage of storage (day 45). Additionally, high-throughput sequencing was used to analyze the microbial community structure and diversity characteristics. The results showed that premium-grade Daqu exhibited the highest fermentation activity (0.41 g/(0.5 g·72 h)) and esterification activity (385.39 mg/(50 g·7 d)), whereas first-grade Daqu had the highest amino acid nitrogen content (4.24 g/kg), and second-grade Daqu exhibited the highest acidity (1.29 mmol/10 g) and acid protease activity (42.54 U/g). The dominant bacterial phyla in all three grades of Daqu were Firmicutes, Proteobacteria, and Actinobacteria, while the fungal community was dominated by the phylum Ascomycota. Bacillus, Thermoascus, and Thermomyces were the dominant genera in these grades of Daqu, with their relative abundances varying among different grades. Linear discriminant analysis effect size (LEfSe) (LDA score ≥ 2, P < 0.05) identified 8 differential bacterial genera and 4 differential fungal genera, among which Bacillus and Thermomyces were the core microbial communities of Daqu at the middle stage of storage. Correlation analysis indicated that dominant genera such as Saccharopolyspora and Aspergillus were significantly correlated with various indicators such as acidity and acid protease activity (P < 0.05). PICRUSt analysis revealed grade-related differences in carbohydrate, amino acid, and nucleotide metabolic pathways. In conclusion, these findings provide a theoretical basis for precise regulation and quality management during the Daqu storage process.