Abstract: Root-knot nematodes (RKN, Meloidogyne spp.) cause soil-borne diseases in food crops, and can lead to a huge crop damage worldwide. It has been demonstrated that application of biogas slurry during planting is an effective method to control diseases caused by RKN. We explored the inhibition effect of biogas slurry on RKN by a soil flooding method to provide scientific basis for a new idea to prevent and control soil-borne diseases. A pot experiment was conducted using soil infected with RKN from a vegetable greenhouse. Four treatments were set: 1) biogas slurry was routinely applied to soil three times during the planting period (BS), with an application rate of NH₄⁺ 50 mg·kg^-1; 2) soil was pretreated by flooding with 70% biogas slurry twice before planting (BSS); 3) soil was covered with mulching films and heated to 45 ℃ (HE) to simulate a conventional smothering process and, 4) soil was untreated (CK). The most obvious inhibition of RKN was BSS treatment, with a control effect of 97.1%. The root-knot index in BSS treatment decreased by 96.9% and 92.9%, respectively, compared with that of HE and BS. However, this method showed a small trend of inhibiting crop growth. Although HE significantly reduced the number of RKN compared with CK, the RKN number rebounded at the later stage (60 d after treatment), and even was higher than that of CK. Taken together, the proportion of herbivores nematode was the highest in CK (mean 81.8%), while bacterivores dominated in the two biogas slurry treatments, BS and BSS (mean 78.3%). Omnivores and carnivores nematode disappeared in soil flooded with biogas slurry, although they reappeared at the destructive sampling period, the relative abundance was still very low. In the pot system, soil flooding with biogas slurry before planting significantly improved the inhibition effect on RKN compared with the application of biogas slurry during planting. This result revealed that the critical period of using biogas slurry to prevent and control RKN is at the larval stage: that is, before nematodes invade plant roots. Further studies are needed under field conditions to study the toxic effects of biogas slurry flooding in plants, and the potential risk of environmental pollution.