In this study, nearly 200 endophytic bacteria were isolated from different part of Huperzia serrata, over 60 bacterium with clear antifungal activity were selected from those cultures. Among them, strain H-6 exhibited the highest antifungal activity which was strongly inhibits the growth of many plant pathogenic fungi such as Sclerotinia scleroliorum, Fusarium graminearumt, Sclerotinia libertiana, Phytophthora capsici Leonia and Sesame fusarium wilt. According to the characteristics of morphology, physiology and biochemistry tests and the comparison of 16S rDNA sequence, the strain H-6 was similar to the Burkholderia. So strain H-6 was identified as Burkholderia sp. H-6. The results also showed that Burkholderia sp. H-6 was markedly different from Burkholderia cepacia that was applied widely in agriculture as antagonistic bacteria. The medium and culture conditions of the strain all were optimized by single factor and orthogonal ex-periments. In the investigation of the culture condition, growth was carried out in a basal medium (potato juice) and gradually supplemented with the various ingredients to be investigated. The major ingredients be-ing investigated included carbon sources and nitrogen sources. The optimal antifungal activity production condition is growth in a medium (potato juice with 2.5% mannitol and 0.1% NaNO3), initial pH 4.0 at 28℃.