The objective of the present study was to assess serum levels of essential trace elements and minerals in non-alcoholic fatty liver disease (NAFLD) patients with and without type 2 diabetes mellitus (T2DM), as well as evaluate their association with metabolic risk biomarkers with adjustment for the prevalence of comorbidities. A total of 260 adults including 77 healthy controls, 107 NAFLD patients without T2DM, and 76 subjects having both NAFLD and T2DM were enrolled in the study. Anthropometric parameters and the prevalence of concomitant diseases were registered. Serum trace element and mineral levels were evaluated using inductively coupled plasma mass-spectrometry. In addition, blood glucose, liver damage markers, and other markers of metabolic risk were assessed using spectrophotometry. The obtained data showed that along with markers of liver damage like ALT and GGT activity, NAFLD patients, especially those having T2DM, are also characterized by hyperglycemia and dyslipidemia. NAFLD patients were also characterized by significantly lower serum Ca, Co, Fe, Mg, and especially Zn levels, being the lowest in NAFLD patients with T2DM. In contrast, a decline in serum Mn and Se concentration was observed only in NAFLD patients without T2DM compared to control subjects. Multiple linear regression analysis with adjustment for various comorbidities revealed a tight inverse association between circulating levels of Zn and metabolic risk markers including ALT activity, glucose, and HbA1c levels, while serum Cu and Se were positively associated with certain parameters. These findings suggest that disturbances of trace element and mineral metabolism are significantly associated with more profound metabolic alterations in NAFLD patients with T2DM. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2025.