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Small intestinal bacterial overgrowth (SIBO) contributes to the formation of an inflammatory environment in various intestinal and extraintestinal diseases. Cytokines that participate in these mechanisms are yet to be examined. Upper gastrointestinal endoscopy with duodenal aspiration was performed in 224 patients. Quantitative cultures of aerobic species were performed, concentrations of interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) were measured, and loads of Escherichia coli, Klebsiella pneumoniae, Methanobevibacter smithii, and Aeromonas spp. were detected via real-time PCR in the duodenal fluid. Analysis showed that the odds ratio (OR) for elevated IL-1β levels was 2.61 (1.06–6.43, p = 0.037) among patients with SIBO compared to patients without SIBO, while there was no significant difference at elevated IL-6 and TNF-α levels between patients with and without SIBO, using ≥10³ cfu/mL as a cut-off. The presence of all three elevated cytokine levels has OR 3.47 (1.06–11.34, p = 0.030) among patients with SIBO. Klebsiella pneumoniae detection was positively related with IL-6 and TNF-α levels, when Methanobevibacter smithii was positively related with IL-1β levels. The presence of SIBO is associated with elevated IL-1β levels in the duodenal fluid. There is a high prevalence of all three proinflammatory cytokine levels elevated (IL-1β, IL-6, and TNF-α) in the duodenal fluid among patients with SIBO.