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The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) underscores the need for better understanding of its complex pathogenesis. Lipid accumulation in hepatocytes is among principal mechanisms contributing to MASLD development. While routine lipid parameters are well studied, the profile of circulating fatty acids in MASLD patients remains less explored. This study aimed to assess relative proportions of individual fatty acids in plasma of MASLD patients and to explore their associations with other biochemical markers of MASLD. Ninety-one patients and 48 healthy individuals were enrolled. The relative proportions of fatty acids in plasma were determined using gas chromatography with FID detection. Proportions of total n-6 polyunsaturated fatty acids (PUFAs) and linoleic acid (LA) in plasma were lower in MASLD patients (p = 0.001 and p = 0.004, respectively), with no differences observed in n-3 PUFAs. Total plasma n-6 PUFAs correlated negatively with body mass index, hepatic steatosis indices, triglyceride concentration and coronary risk index. Decreased prevalence of n-6 PUFAs in plasma was independently associated with higher odds of MASLD (OR = 0.769; CI: 0.611–0.968; p = 0.025). Our findings indicate an altered circulatory fatty acid distribution in MASLD, characterized by a reduced amount of n-6 PUFAs, particularly LA, which may have significant implications for the prevention and treatment of MASLD.