Abstract: To clarify the main pathogens causing rhizome diseases in tobacco float seedling in Chenzhou City, Hunan Province in recent years,and screen effective fungicides, pathogens were isolated by tissue isolation method. Their taxonomic status and pathogenicity were determined through morphological observation, molecular biological identification (rDNA–ITS and COX1 sequence analysis) and Koch’s postulates verification. Indoor toxicity assays were carried out by the mycelial growth rate method, and effective fungicides were screened combined with pot experiments.The results showed that the pathogen responsible for tobacco root rot was Aphanomyces mitsuba. Indoor toxicity tests indicated that Aphanomyces mitsuba exhibited high sensitivity to 0.3% eugenol soluble concentrate (SL) and 58% metalaxyl-mancozeb wettable powder (WP), with EC50 values of (2.387 3±0.338 0) μg/mL and (3.599 7±0.481 3) μg/mL, respectively. In pot control experiments, 58% metalaxyl-mancozeb WP exhibited excellent control efficacy against this disease, with a relative control efficacy of (55.91±2.62)%.