In this study, we investigated the use of nematode-infected Pinus massoniana chips (NPC) as the main ingredient in Pleurotus abieticola substrate. The effects of different substrate formulas on nutritional parameters, including total sugars, polysaccharides, proteins, lipids, amino acids, and fatty acids were assessed. The results indicated that NPC was suitable for P. abieticola cultivation. However, the addition of certain amounts of corncobs (CC) and cottonseed hulks (CH) improved the yield. Substrate T5 (45% NPC, 6% CC, and 27% CH) had the greatest yield (121.38 g per bag), 34.56% greater than the yield of the control (78% poplar chips), which was 79.43 g per bag. Across the 11 substrate formulas tested, the total sugar, polysaccharide, crude protein, and crude lipid contents were 16.60-28.90%, 2.71-3.73%, 36.49-45.42%, and 1.03-4.34%, respectively. On all substrates, the fruiting bodies contained 17 amino acids, primarily glutamine (2.42-4.11%), followed by proline (2.56-3.73%), leucine (2.09-3.19%), phenylalanine (1.56-2.61%), and glycine (1.76-2.55%). The fruiting bodies contained 12 fatty acids, of which linoleic acid was the most abundant (82.36%-84.03%), followed by palmitic acid (6.42%-6.89%) and oleic acid (5.50%-7.34%). The fatty acid content was closely associated with the NPC content, which might indicate that NPC promoted fatty acid accumulation. Thus, NPC represents a new substrate suitable for P. abieticola cultivation.