The effects of breed and diet containing different types of silages on meat quality parameters and the fatty acid profile of m. longissimus lumborum (MLL) were evaluated in Czech Fleckvieh (CF), Charolais (CH) and Charolais x Czech Fleckvieh (CH x CF) bulls. The animals were fed two mixed diets: MS (based on maize silage) and LCS (based on legume-cereal mixture silage and lucerne silages) with different concentrations of dietary energy and fatty acids. The MLL from CH bulls had the lowest content of dry matter, less protein, and lighter meat compared to the CF. The CH bulls exhibited higher PUFA n-3 and lower MUFA compared to the CF, with the CHxCF being intermediate. The LCS diet enhanced the proportions of PUFA and PUFA n-3 and reduced MUFA. The replacement of maize silage with the legume-cereal mixture and lucerne silages in the diet reduced the concentration of intramuscular fat and improved its fatty acid profile from the human nutrition perspective.