The aim of the study was to compare the skeletal muscles (longissimus lumborum – LL vs. semitendinosus – ST) of Polish Holstein-Friesian bulls in terms of their chemical composition, fatty acid proportions and the degree to which they meet standards for selected nutrients. The proximate composition, that is, the content of water, ash, crude protein, fat and collagen, was determined in the muscle samples collected. The calorific value of 100 g of meat was calculated based on the total protein content and intramuscular fat, using energy equivalents: 4 kcal for protein and 9 kcal for fat. The fatty acid profile (FA) was determined by gas chromatography (GC-FID), and the following groups were distinguished: saturated fatty acids (SFA), monounsaturated fatty acids in the cis (MUFA cis) and the trans configuration (MUFA trans), as well as polyunsaturated fatty acids (PUFA). On the basis of the content of individual fatty acids, the ratio of PUFA/SFA and n-6/n-3 was calculated. In addition, the following indices were calculated: atherogenic index (AI), thrombogenic index (TI), saturation index (S/P), as well as nutritional value (NV) and the ratio of hypo- and hypercholesterolaemic fatty acids (h/H). The results obtained for fatty acids were expressed as 1) percentage of individual fatty acids in total FAs quantified and 2) as quantity (in mg) per 100 g of fresh meat. Statistical calculations were performed using Statistica software ver. 13. Differences between the muscles were verified with the t-test for independent samples, assuming significance levels of p ≤ 0.05 and p ≤ 0.01. The muscle type was found to significantly influence the fatty acid (FA) profile for four of the 24 FAs identified. Compared to ST, the LL muscle had a significantly higher share of C18:0 (18.95% vs. 16.55%; p ≤ 0.01) and CLA (0.38% vs. 0.29%; p ≤ 0.05) and significantly lower percentages of C16:1 c9 (3.17% vs. 3.74%; p ≤ 0.05) and C18:1 c9 (36.02% vs. 38.65%; p ≤ 0.05). With regard to particular groups of fatty acids, the LL muscle had significantly higher percentages of SFA (53.20% vs. 50.71%, p ≤ 0.05), PUFA (4.20% vs. 3.49%, p ≤ 0.05) and n-6 (2.69% vs. 2.10%, p ≤ 0.01) and a significantly lower percentage of cis MUFA (40.73% vs. 43.96%; p ≤ 0.01). The average amount of fat in 100 g of muscle tissue was 693.30 mg for SFA, 560.81 mg for MUFA cis, 54.74 mg for PUFA (including 33.87 mg of n-6 and 16.23 mg of n-3) and 24.63 mg for MUFA trans. The beef can be classified as a secondary source of fatty acids. The coverage of the recommended intake ranged from 2.5% to 3.8% for SFA, from 1.1% to 1.9% for MUFA, from 0.29% to 1.8% for n-3 acids, and was smaller than 1% for PUFA and n-6. The amount of EPA and DHA satisfied from 0.66% to 1.3% of the minimum daily requirement, while the amount of trans FA ranged from 0.9% to 1.4% of the maximum daily level. It is worth noting that 100 g of the beef provided on average 102.53 kcal, 1.46 g of fat and 22.34 g of protein, which cover, respectively, 5.1% of the daily calorie requirement, 1.9-3.3% of the fat requirement, and as much as 30-45% of the protein requirement for adults. Although the ST muscle, because of its 40% lower fat content and 2% lower protein content, was less caloric than the LL muscle, it also contained fewer biologically active FAs, including CLA, VA, ALA, LA, AA, EPA and DPA. Irrespective of the differences shown, the meat can be classified as a low-calorie and high-protein product, as 87% of its energy value came from protein.