Recent research highlights a potential link between metabolic dysfunction-associated steatotic liver disease (MASLD) and intestinal barrier dysfunction. Increased intestinal permeability (IP) may facilitate the translocation of bacteria, endotoxins (e.g., lipopolysaccharides [LPS]), and pathogen-associated molecular patterns into the portal venous system, fostering a pro-inflammatory environment and contributing to liver inflammation. This study aimed to identify correlations between intestinal barrier biomarkers (occludin, LPS, and intestinal-type fatty-acid-binding proteins [I-FABP]) and MASLD. A single-center prospective cross-sectional study was conducted, including 72 MASLD patients and 68 healthy controls. Fibroscan-controlled attenuation parameter (CAP) was performed in all subjects. Blood samples were analyzed for biochemical parameters, and serum levels of occludin, LPS, and I-FABP were measured using the ELISA method with the Human occludin, LPS, and I-FABP ELISA Kit test systems (FineTest, Wuhan, China). LPS and I-FABP levels were significantly higher in MASLD patients compared to controls, with the highest LPS levels observed in the diabetic MASLD subgroup. Occludin levels showed no statistically significant differences between groups. All 3 biomarkers were positively correlated with BMI, with the highest levels in obese subjects. LPS was positively correlated with CRP levels. Using Fibroscan-CAP, we found a positive correlation between LPS and both liver stiffness and CAP score, as well as between I-FABP and liver stiffness. MASLD patients exhibit increased IP, with enterocyte injury present irrespective of diabetes status, though more pronounced in diabetic MASLD. Occludin does not appear to be a reliable biomarker for evaluating intestinal barrier function in MASLD. Obesity is linked to elevated biomarkers, suggesting an association between increased IP and obesity. I-FABP and LPS may serve as noninvasive biomarkers for assessing hepatic fibrosis and steatosis in MASLD patients. Notably, LPS, given its correlation with elevated CRP levels, could be utilized as a marker of disease progression and severity.