The antimicrobial effect of solvent extracts from the rhizome of a thick-stemmed wood fern (Dryopteris crassirhizoma) was evaluated and its phloroglucinol components, flavaspidic acids PB and AB. Flavaspidic acids PB and AB were isolated from the D. crassirhizoma rhizomes by methanol extraction, followed by silica gel and Sephadex LH-20 column chromatography. The chemical structures were characterized by spectral techniques, including ESI-MS, UV, (1)H- and (13)C-NMR spectrum analysis. When the antimicrobial activity of the extracts and compounds was tested by the paper disc method, the extracts and compounds were highly active against Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus KCTC 1928 (a MRSA bacterium), Streptococcus mutans and Bacillus subtilis. The extracts and compounds were not active against fungi and chlorella. Our study revealed that the antibacterial activity of samples from D. crassirhizoma was mainly related to the flavaspidic acids.