Thirty-three fungal strains were isolated from the healthy tissue of marine sponge by three different media. They were identified by morphology characteristics analysis and rDNA ITS sequence analysis．The species of each strain was determined, and the dominant fungi of Aspergillus and Penicillium were found. Among them, M3 was Aspergillus versicolor. The antimicrobial activity of the metabolites of the symbiotic fungi of marine sponge was studied by 96-well plate method with the standard strain of Escherichia coli. The results showed that the secondary metabolites of seven strains of symbiotic fungi had certain antimicrobial activity. The inhibition rate of M3 on Escherichia coli was (56.66±2.28)%, followed by M1, with the inhibition rate was (51.23±6.24)%. Both of them of higher than 100 mmol/L penicillin.