A new fructofuranoside glycerol, dryoptkirbioside (1), along with fourteen known compounds (2‐15), was isolated from the MeOH extract of Dryopteris kirbi rhizomes by silica gel column chromatography, Sephadex LH‐20 column chromatography, and semipreparative HPLC. The structure of the new compound was determined by analyses of its spectroscopic data including nuclear magnetic resonance (NMR), and high‐resolution electrospray ionisation mass spectrometry (HRESI‐MS) and chemical conversions. The n‐hexane‐soluble portion and the EAFA fraction showed strong activities against lung (A549), breast (MCF‐7), and cervical (HeLa) human cancer cell lines (IC50 values ranging from 4.0 to 8.8 µg/mL). Aspidinol P (5) and aspidinol B (6) exhibited moderate to low cytotoxicity on the three cell lines (IC50 values ranging from 20.4 to 58.7 µM). The MeOH extract and n‐hexane‐soluble portion had excellent activities against Staphylococcus aureus and Bacillus subtilis (MICs 11.7 and 23.4 µg/mL), whereas the EtOAc‐ and n‐BuOH‐soluble portions were significantly active on S. aureus (MICs 46.9 and 93.8 µg/mL). The main fractions EAFB, EAFC and nBFB displayed excellent activity against S. aureus (MICs 11.7 and 23.4 µg/mL). Aspidinol B (6) had significant activity, while aspidinol P (5) was moderately active against S. aureus and B. subtilis (MICs 42.0 and 89.5 µM).