Background and Aims The occurrence of infection during the course of acute pancreatitis (AP) is a major factor leading to high mortality rates in critically ill patients. However, severe infection-related complications often manifest more than 2 weeks after onset, and the positive rate of fluid bacterial culture in the early stages of the disease is low, making early antibiotic use lacking in guidance. Blind prophylactic use of antibiotics may face the risk of fungal and antibiotic-resistant infections, exacerbating the condition. Previous studies have found that bacterial infections already exist in the early stages of the disease, and intestinal flora migrate to the pancreas through various pathways to participate in the occurrence and development of the disease. Fluid bacterial culture may provide reference basis for early antibiotic use, but currently, there are few reports on relevant pathogenic studies. Therefore, this study was performed to seek evidence of early infection in AP through bacterial culture of pancreatic juice, to provide reference for anti-infective treatment of AP.Methods AP patients who underwent bacterial culture of pancreatic juice in the Hepatobiliary Surgery Department of Ningxia Medical University General Hospital from January 1, 2019 to June 30, 2020 were reviewed. The results of bacterial culture and clinical data of patients were recorded and analyzed.Results A total of 156 patients were included in the study, of which 64 (41.03%) had positive fluid bacterial cultures. A total of 94 bacterial strains were cultured, with gram-negative bacteria (58.51%) being the most common, followed by gram-positive bacteria (38.30%) and fungi (3.19%). The distribution and composition of pancreatic fluid bacteria in biliary AP, hyperlipidemic AP, and idiopathic AP were similar. The incidence of complications, APACHE Ⅱ score, and levels of inflammatory markers in patients with positive pancreatic fluid bacterial culture were significantly higher than those in patients with negative culture, and the duration of fever was also significantly longer in the positive culture group than in the negative culture group (all P<0.05). Among the 21 patients who developed infected pancreatic necrosis (IPN) in the later stage, 19 cases had early positive fluid cultures, and 9 IPN patients underwent percutaneous drainage, with a 100% (9/9) consistency between the drainage fluid culture and the early pancreatic fluid culture results. In the population of patients with positive pancreatic fluid bacterial cultures, non-infected patients were significantly older than infected patients, and the proportion of non-infected patients with biliary AP was also significantly higher (both P<0.05). In elderly patients with biliary AP, there was no significant difference in the incidence of complications and levels of inflammatory markers between the positive and negative fluid culture groups (all P>0.05).Conclusion The bacteria in early pancreatic fluid of AP patients are mainly intestinal flora and are related to the severity of the disease. Targeted use of antibiotics may have a positive impact on the outcome, but it should be evaluated and used with caution in elderly patients with biliary AP.