Cyst nematodes are major crop pests, causing severe economic losses for farmers. Among the pest management alternatives, the suicide hatching strategy, which aims to mislead the nematode by using host specific root exudates, has already proved its efficiency on several cyst nematodes including Heterodera carotae. However, previous studies have highlighted several constraints, one of which being the impact of soil microbial communities on suicide hatching efficiency. Indeed, plants secrete root exudates to recruit beneficial microorganisms which provide major ecological functions and help the plants to face biotic and abiotic stresses. Root exudates are thus a signal recognized by microorganisms in the soil but also by cyst nematodes to begin their parasitic cycle. In this study, we assessed the efficiency of the suicide hatching strategy depending on its positioning in the agronomic system. Indeed, potential variations in efficiency are expected due to the specific composition of the soil microbiota selected by the different plant species likely to be cultivated in rotation with the carrot. To do so, we first grew for 3 cycles of 3 weeks, under controlled conditions, three plants (leek, turnip and barley) usually cultivated with the carrot, in 2 distinct soils (sandy and muddy soil). Bags containing 12 H. carotae cysts were added to these microbial-enriched soils before applying the biocontrol solution (carrot root exudate) at two doses and in the presence or absence of the corresponding plant to compare the hatching efficiency of an application of the product during or following the cultivation. At the end of the experiment, we calculated the hatching rate and analysed the soil microbiota composition and structure using a metabarcoding approach targeting bacteria and fungi. We clearly demonstrated a specialisation of the soil microbiota depending on the rotation plant at the end of the 3 cycles of growth. Moreover, in the sandy soil, we observed clear patterns with a greater hatching rate when the plant is present compared to the bare soil and the highest rate when the barley is cultivated. Further statistical analyses should enable us to attribute or not the hatching rate variations to the microbial communities and to identify favourable and/or unfavourable microorganisms specific to the barley or to the leek, respectively. Taken together, our results will allow us to propose the best positioning of the biocontrol product in the technical itinerary by considering the soil microbiota.