As an cultivated aquatic vegetable, the long-term continuous monocropping of water oat results in the frequent occurrence of diseases, the deterioration of ecological system and decreased quality of water oat. In this study, real-time quantitative PCR (qPCR) and Illumina high-throughput sequencing were used to determine the dynamic changes in bacterial and fungal communities in rhizosphere soil under continuous cropping of water oat for 1, 5, 10, 15 and 20 years (Y1, Y5, Y10, Y15 and Y20), and soil properties and enzyme activities were also determined. Results showed that the contents of soil organic carbon (SOC), total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), available phosphorus (AP) and the activities of four soil enzymes increased in Y5 and Y10 and then decreased in Y15 and Y20. Spearman correlation analysis identified SOC, TN, AP and AN as the main factors that affect the four enzyme activities. The qPCR results showed that there was no significant difference in bacterial abundance between the different planting years, while the fungal abundance first increased and then decreased. The long-term continuous planting of water oat (Y15 and Y20) significantly reduced the operational taxonomic unit numbers and the Shannon, Chao1, and ACE indices of rhizosphere bacteria and fungi. The bacterial and fungal community compositions were markedly affected by the continuous planting year. The relative abundances of Bacteroidetes and Firmicutes decreased significantly in Y10 and Bacteroidetes increased significantly in Y15. Relative abundances of dominated Mortierellomycota and Ascomycota phyla increased with the continuous cropping years, while Rozellomycota presented the opposite trend. The AK, AN, and SOC were the main factors that changed the bacterial community, while AK and AP significantly shifted the fungal community. Thus, long-term continuous planting of water oat resulted in the deterioration of soil nutrients and microbial communities. The results provided a reference for the remediation of soil under continuous water oat planting and sustainable development of water oat industry.