ObjectivesThis study aimed to identify the bacterial composition in the pancreatic fluid of severe and critical acute pancreatitis (SAP and CAP) patients.MethodsA total of 78 pancreatic fluid samples were collected from 56 SAP and CAP patients and analyze using aerobic culture and16S rRNAgene next-generation sequencing. The clinical data of the patients were obtained from the electronic medical records.ResultsAmong the total 78 samples,16S rRNAgene NGS identified a total of 660 bacterial taxa, belonging to 216 species in 123 genera. The dominant aerobic bacteria includedKlebsiella pneumoniae,Acinetobacter baumannii, andEnterococcus faecium, while the dominant anaerobic bacteria includedBacteroides,Dialister invisus, andOlsenella uli. As compared to aerobic culturing, 95.96% (95/99) of the aerobic cultured bacteria were detected using the16S rRNAgene NGS.ConclusionThe pancreatic infections in SAP and CAP patients might originate not only from the gut but also from the oral cavity and airways as well as related environments. Dynamic analysis of bacterial profile and abundance showed that some bacteria with low abundance might become the main pathogenic bacteria. There were no significant differences in the bacterial diversity between SAP and CAP.