Metabolic dysfunction-associated steatotic liver disease (MASLD) is a serious health problem, and recent evidence indicates that gut microbiota plays a key role in its development. It is known that 2-oleoyl glycerol (2-OG) produced by the gut microbiota is associated with hepatic fibrosis, but it is not known whether this metabolite is involved in the development of hepatic steatosis. The aim of this study was to evaluate how a high-fat-sucrose diet (HFS) increases 2-OG production through gut microbiota dysbiosis and to identify whether this metabolite modifies hepatic lipogenesis and mitochondrial activity for the development of hepatic steatosis as well as whether a combination of functional foods can reverse this process. Wistar rats were fed the HFS diet for 7 months. At the end of the study, body composition, biochemical parameters, gut microbiota, protein abundance, lipogenic and antioxidant enzymes, hepatic 2-OG measurement, and mitochondrial function of the rats were evaluated. Also, the effect of the consumption of functional food with an HFS diet was assessed. In humans with MASLD, we analyzed gut microbiota and serum 2-OG. Consumption of the HFS diet in Wistar rats caused oxidative stress, hepatic steatosis, and gut microbiota dysbiosis, decreasing α-diversity and increased abundance, which increased 2-OG. This metabolite increased de novo lipogenesis through ChREBP and SREBP-1. 2-OG significantly increased mitochondrial dysfunction. The addition of functional foods to the diet modified the gut microbiota, reducing and 2-OG levels, leading to a decrease in body weight gain, body fat mass, serum glucose, insulin, cholesterol, triglycerides, fatty liver formation, and increased mitochondrial function. To use 2-OG as a biomarker, this metabolite was measured in healthy subjects or with MASLD, and it was observed that subjects with hepatic steatosis II and III had significantly higher 2-OG than healthy subjects, suggesting that the abundance of this circulating metabolite could be a predictor marker of hepatic steatosis.