Biofumigation, although a well-known method, is still controversially debated as a management strategy for plant-parasitic nematodes (PPN). Its controlling effect is attributed to the production of isothiocyanates (ITCs) following the action of myrosinase on glucosinolates (GSLs). Different ITCs are formed from different GSLs, depending on the plant species. To better understand the potential of ITCs, eight cultivars from three Brassicaceae species were investigated as biofumigation crops to control the root knot nematode Meloidogyne hapla. Since results were inconsistent, the nematicidal effect of selected ITCs were further evaluated in vitro. Based on its nematicidal potential, allyl ITC (AITC) was specifically investigated under different soil:sand compositions. A significantly lower nematicidal activity was observed in soil compared to sand. AITC was also evaluated as an additive to the biofumigation in a greenhouse trial. Its supplementation to the biofumigation process with Brassica juncea cv. Terrafit controlled M. hapla, while no control was observed using Raphanus sativus cv. Defender. Thus, the success of biofumigation seems to be strongly dependent on the soil characteristics and the ITC produced during the biofumigation process. Therefore, the supplementation of AITC in combination with the right cover crop can improve the biofumigation process to control M. hapla.