The effects of breed and diet containing different types of silages on meat quality parameters and fatty acid profile of m. longissimus lumborum (MLL) were evaluated in a total of 30 Czech Fleckvieh (CF), Charolais (CH) and Charolais × Czech Fleckvieh (CH × 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 (P < 0.01), less protein (P < 0.01) and lighter meat (P < 0.01) compared to the CF. The extensive LCS diet reduced dry matter (P < 0.01) and intramuscular fat (P < 0.01) and increased the content of hydroxyproline (P < 0.05). The CH bulls exhibited higher PUFA n-3 (P < 0.05) and lower MUFA (P < 0.05) compared to the CF, with the CH × CF being intermediate. The LCS diet enhanced the proportions of PUFA (P < 0.05) and PUFA n-3 (P < 0.001) and reduced MUFA (P < 0.001). In conclusion, both breed and diet affected the meat quality and fatty acid profile of the intramuscular fat of the bulls. 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.