Nineteen strains of endophytic fungi were isolated from the stems, roots and leaves of Paulownia fortune, their antimicrobial activity against three tested microorganisms were investigated by using the agar diffusion method and the paper disc method. Identification of these strains were determined by their morphological features and phylogenetic analysis based on internal transcribed spacer of rRNA gene sequence comparison. The results showed that endophytic fungi of JSD-8, JM-1 and JM-10 had remarkable antimicrobial activities to Bacillus subtilis CGMCC 1.769, Escherichia coli CGMCC 1.1103 and Candida albicans ATCC 10123, respectively. Fungal morphological and molecular identification demonstrated that JSD-8, JM-1 and JM-10 represented Gibberella moniliformis, Alternaria longipes and Penicilium thomii, respectively.