Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by the intracellular lipid accumulation in hepatocytes. Excess caloric intake and high-fat diets are considered to significantly contribute to MASLD development. To evaluate the hepatic and serum fatty acid (FA) composition in patients with different stages of MASLD, and their relationship with FA dietary intake and MASLD-related risk factors. This was a case-control study in patients with obesity undergoing bariatric surgery at a university hospital between January 2020 and December 2021. Participants were distributed in 3 groups: no MASLD (n = 26), steatotic liver disease (n = 33), and metabolic dysfunction-associated steatohepatitis (n = 32). Hepatic and serum FA levels were determined by gas chromatography-mass spectrometry. Nutritional status was evaluated using validated food frequency questionnaires. The hepatic expression of genes involved in FA metabolism was analyzed by reverse transcription quantitative polymerase chain reaction. The hepatic, but not serum, FA profiles were significantly altered in patients with MASLD compared with those without MASLD. No differences were observed in FA intake between the groups. Levels of C16:0, C18:1, and the C18:1/C18:0 ratio were higher, while C18:0 levels and C18:0/C16:0 ratio were lower in patients with MASLD, being significantly different between the 3 groups. Hepatic FA levels and ratios correlated with histopathological diagnosis and other MASLD-related parameters. The expression of genes involved in the FA metabolism was upregulated in patients with MASLD. Alterations in hepatic FA levels in MASLD patients were due to enhancement of de novo lipogenesis in the liver.