Abstract OBJECTIVE To establish an environmental microbial identification information database for a high-risk production area of sterile formulations in pharmaceutical enterprises using various microbial identification techniques. METHODS For four consecutive quarters, microorganisms were collected from intermediates, pharmaceutical water, clean spaces, personnel and equipment surfaces, and identified using VITEK biochemical identification, MALDI-TOF MS protein identification, and 16S rRNA/ITS gene identification techniques. Combined with microbial morphology and source information analysis, a corresponding microbial identification information database was established. RESULTS A total of 89 bacterial and 5 fungal identification results were collected and related morphological analysis was conducted. Among the 94 collected strains, Gram-positive Cocci and Gram-positive Bacillus accounted for 60.6%, which was the common contaminating bacteria in clean areas; 46 strains of microorganisms were collected in the B-level environment, with Staphylococcus being the main dominant genus, accounting for 45.7%, followed by Bacillus(17.4%), Micrococcus(6.5%), and Yeast(4.3%), one strain of Burkholderia cepacia was also isolated in the B-level environment; 29 strains of microorganisms were collected from pharmaceutical water, mainly Gram-negative bacteria(58.6%). CONCLUSION This study establishes a microbial identification information database that integrates biochemical, protein, and gene identification results, and conduct relevant analysis to provide a basis for pharmaceutical enterprises to carry out risk control, strengthen the management of unacceptable microorganisms, and conduct microbial data deviation surveys and traceability analysis; enterprises can also use the identification results to establish an environmental microbial map, control and eliminate microbial pollution in a targeted manner, improve the level of sterility assurance, and ensure the quality and safety of drugs.