Tomato crops are affected in Mediterranean cold-greenhouse agrosystems by soilborne diseases, such as root-knot nematodes (Meloidogyne spp.), which represent a serious problem leading to losses in production. Agroecological soil management based on biocontrol agents and natural compounds has had increased grower interest in order to reduce chemical residues in the produce and to adopt environmentally friendly farming methods. In this frame, we evaluate and validate soil biofumigation by the use of glucosinolate (GLS) compounds. Among them, sinigrin showed biocontrol activities against several pests and diseases via nematotoxic action. Among the Brassicaceae species rich in sinigrin, we chose Brassica macrocarpa Guss. (BM) because its leaves show 90% of all GLSs, and we could better estimate the action of this single GLS. Different dosages of BM leaf flour, containing 200 to 300, 350, 400, 450, and 650 μmol m−2 of sinigrin, were inserted into soil already infected by Meloidogyne spp. for evaluating their effects on tomatoes grown in cold greenhouses in comparison to absolute control (CTRL) and to the chemical one, Vydate 5G® (CCTRL). The root disease index, caused by nematode attack, was the highest in CTRL, and a reduction of about 50% was observed with the 300 to 650 μmol m−2 sinigrin dosage. The CCTRL showed twofold marketable yield increase, and a fourfold increase was found in 650 μmol m−2 of sinigrin dosage, in comparison to the CTRL. Biofumigant applications improved tomato plant growth and development, and fruit quality, significantly for dry matter and soluble sugars (°Brix). BM leaf flour inserted into the soil, at a dose of 300 μmol m−2 of sinigrin, showed similar effects to the CCTRL on root disease index, root weight, and marketable yield. Data showed the nematotoxic effect of sinigrin for the biocontrol of Meloydogine spp. by the use of B. macrocarpa leaves, very rich in this GLS compound, which represents a new tool for agroecological soil management and for organic farming.