The lotus (Nelumbo nucifera Gaertn.) is widely cultivated in Asia, but a blackening disease in the lotus tuber, called “kurokawa-senchu-byo”, is a serious problem caused by the Hirschmanniella diversa Sher plant-parasitic nematode. To effectively control the disease, we must elucidate the blackening mechanisms; therefore, in this study, we performed a soil chemical analysis and an evaluation of the disease level in the lotus cultivation fields, identified the chemical components of the black spots on the lotus surface, and performed a 16S rRNA gene-based microbial community analysis of the black spots. Using linear regression analysis, a positive linear relationship with a strong correlation between the damage index values and fertilizer components such as P2O5 was observed. As a result of scanning electron microscopy and energy-dispersive X-ray spectroscopy analysis, phosphorus (P) and iron (Fe) were found to be concentrated in the black spots of the lotus tubers. Furthermore, we found that the concentrations of P and Fe in the black spots were 1.5- and 2.7-fold higher, respectively, than those found in the healthy parts of the lotus tubers. A 16S rRNA gene analysis revealed that dissimilatory Fe(III)-reducing bacteria (DIRB) were predominant in the black spots, suggesting that these bacteria are important to the formation of P and Fe compounds in the black spots.