BACKGROUND: Infectious pancreatic necrosis (IPN) is associated with the prognosis of acute pancreatitis (AP). The correlation between microbial diversity in the peripancreatic region and AP severity remains unclear. This study investigates whether pathogen profiles in peripancreatic necrosis differ between IPN patients with and without organ failure (OF), using 16 S ribosomal RNA (16 S rRNA) and internal transcribed spacer (ITS) sequencing. METHODS: Patients with acute pancreatitis meeting clinical diagnostic criteria for IPN were prospectively enrolled. These patients were subsequently divided into an OF group and a non-OF group. Peripancreatic necrotic fluid samples were collected via percutaneous drainage and subjected to 16 S rRNA and ITS sequencing. RESULTS: A total of 28 patients with suspected infected pancreatic necrosis (IPN) were included, with 14 patients in the OF group and 14 in the non-OF group. No significant difference was observed in bacterial alpha diversity between the OF and non-OF groups. However, beta diversity showed significant differences according to Welch's t test (P < 0.001), Adonis analysis (P = 0.037) and Anosim analysis (P = 0.032). The relative abundance of Klebsiella significantly differed between the two groups (2.79% vs. 34.14%, P = 0.0093) and correlated with the length of hospital stay (r = 0.422, P = 0.025). CONCLUSION: The peripancreatic microbial profile is associated with the severity of acute pancreatitis, with Klebsiella being the dominant microflora in patients with IPN combined with OF.