Abstract: To identify fungi with biocontrol potential against tobacco black shank, endophytic fungi were isolated from Bletilla striata. An antagonistic strain with the highest inhibition rate against the pathogen (Phytophthora nicotianae) of tobacco black shank was screened by plate confrontation cultivation. The antimicrobial spectrum of the antagonistic strain was determined and identified by morphological and phylogenetic analyses. The production of amylase and synthesis of indole-3-acetic acid (IAA) and siderophores by the strain were tested, and its relative control efficacy against tobacco black shank was evaluated through pot experiments. The results showed that strain J41, isolated from the roots of Bletilla striata, had an inhibition rate of 81.21% against the mycelial growth of Phytophthora nicotianae. Strain J41 also inhibited the mycelial growth of Fusarium fujikuroi, Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum, and Alternaria solani. The strain J41 was identified as Talaromyces flavus and could produce amylase and synthesize IAA and siderophores. Strain J41 exhibited a relative control efficacy of 66.71% against tobacco black shank, which was not significantly different from that of 58% metalaxyl·manganese zinc wettable powder (P > 0.05).