Abstract: To clarify the main pathogen causing rhizome diseases of tobacco seedlings in floating system in Chenzhou City, Hunan Province in recent years and to screen effective control agents, the pathogen was isolated by the tissue isolation method, and its 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 assay was carried out by the mycelial growth rate method, and effective control agents were screened by 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 good control effects against the disease, with a relative control efficacy of (55.91±2.62) %.