Abstract: Dry rot, predominantly caused by the Fusarium genus fungus Fusarium oxysporum, is a major disease affecting Fritillaria thunbergii Miq., an important medicinal plant in Zhejiang Province. The aim of this study was to select potential biocontrol fungi and chemical agents from indigenous medicinal plants in Zhejiang, with the goal of developing innovative biological control measures against dry rot. Utilizing PCR amplification and sequencing of six gene sequences, including the internal transcribed spacer (ITS) region, coupled with BLAST homology comparison and phylogenetic tree construction, three potentially antagonistic fungi were accurately identified: Trichoderma hamatum, Penicillium rubens, and Irpex lacteus. Assessment of the in vitro antifungal efficacy of potentially antagonistic fungi and existing laboratory strains Chryseobacterium aquaticum, Bacillus subtilis, and Bacillus licheniformis against F. oxysporum using the agar confrontation method and the agar dilution method. The results from the five-point confrontation test showed that five days after inoculation, the average inhibition rates of the six potentially antagonistic microorganisms were 76.48%, 50.17%, 58.28%, 48.88%, 66.26%, and 23.86%, respectively. In the two-point confrontation test, the average inhibition rates were 47.92%, 50.72%, 34.22%, 21.07%, 17.18%, and 13.16%, respectively. The agar dilution method demonstrated that the inhibitory effects of the six potentially antagonistic microorganisms increased with the concentration of sterile fermentation broth within the range of 1% to 20%, with maximum inhibition rates of 22.91%, 21.49%, 23.61%, 46.06%, 3.93%, and 2.67%, respectively. The chemical fungicide screening results showed that the EC₅₀ values of chlorothalonil, phenamacril, pyraclostrobin, and fludioxonil against F. oxysporum were 1.14, 4.92, 0.15, 0.25 μg/mL (spore germination) and 3.60, 11.07, 4.02, 0.13 μg/mL (mycelial growth), respectively. The results indicated that six biocontrol agents showed significant inhibitory activity, particularly T. hamatum, P. rubens, I. lacteus, and C. aquaticum. Fludioxonil exhibited the best antifungal activity among the chemical fungicides tested.