Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (FOC) is a worldwide soilborne disease that causes serious economic losses every year. Effective ways of preventing the disease are still not available. In this study, biological soil disinfestation (BSD) with incorporation of various organic matters under flooding conditions combined with plastic mulching was applied to suppress FOC and prevent banana Fusarium wilt disease. The biological properties of the soil during the BSD process were investigated using quantitative real-time PCR and denaturing gradient gel electrophoresis. The results showed that the values of soil redox potential significantly decreased by >800 mV in the treatments that incorporated rice straw and bagasse under flooding conditions compared with untreated soil. The lowest soil pH (5.36) and a significant decline in the FOC population to 2.79 % of untreated soil were obtained in flooded soil with the highest amount of rice straw. Incorporation of bagasse, pig manure, and lime also significantly decreased the populations of FOC, but were less effective than rice straw. Application of rice straw under flooding conditions significantly increased soil bacterial diversity, and the increased bacteria were considered to have an antagonistic effect on FOC. In 2013 field experiment, a wilt disease control efficiency of 82.3 % was obtained in flooded soil incorporated with 0.5 % (w/w) rice straw compared with untreated soil, and the control efficiency of BSD was further confirmed in 2014 field experiment. These results indicated that BSD can reduce FOC populations in soil, ameliorate soil microbial communities, and reduce the occurrence of banana Fusarium wilt disease.