Fusarium wilt is an increasingly serious disease of watermelon that reduces crop productivity. Changes in microorganism populations and bacterial and fungal community structures in rhizosphere soil of watermelon cultivars resistant or susceptible to Fusarium oxysporum f. sp. niveum were investigated using a plate culture method and PCR-DGGE analysis. Plate culture showed that populations of culturable bacteria and actinomycetes were more abundant in the rhizosphere of the resistant watermelon cultivar than the susceptible cultivar, but the fungi population had the opposite pattern. Populations of Penicillium , Fusarium , and Aspergillus were significantly lower in the resistant cultivar than the susceptible cultivar at the fruiting and uprooting stages (p < 0.05). Pattern matching analysis generated the dendrogram of the DGGE results indicating the relatedness of the different resistant watermelon cultivars and their corresponding rhizosphere microbial communities. Further sequencing analysis of specific bands from DGGE profiles indicated that different groups of bacteria and fungi occurred in the rhizosphere of different watermelon cultivars. Our results demonstrated that plant genotype had a significant impact on soil microbial community structure, and the differences in the rhizosphere microbial community may contribute to the differences in resistance to F. oxysporum f. sp. niveum.