Zostera marina is a submerged monocotyledonous plant, and it is one of the main seagrasses that constitutes the seagrass bed. It^ʼs a good way to study the endophytic actinomycetes associated with Zostera marina for the discovery of marine microbial resource and novel bioactive natural products. Culture-dependent method was used to isolate endophytic actinomycetes associated with Zostera marina from the Swan Lake of Rongcheng, Weihai, Shandong Province. Seven types of media including 2216E were used for actinomycete isolation. Antimicrobial activity of the crude extracts of all isolated strains was determined using filter paper method against seven indicator bacterial strains including Staphylococcus aureus,Candida albicans and Vibrio parahaemolyticusthe, etc. A total of 62 endophytic actinomycetes was isolated from Zostera marina, and the endophytic actinomycetes isolated on the RO medium were rich of strains and abundant. Of the 62 strains, 48 strains (77.42% of strains) showed antimicrobial activities against at least one of seven indicator strains. There were 36 strains (58.06% of strains) against two or more kinds of indicator bacteria. Isolated strains exhibited higher antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) than against Gram-negative bacteria. Our results suggest that endophytic actinomycetes associated with Zostera marina have good diversity and antimicrobial activity, which may be a new source for the discovery of marine antibiotics.