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Ethnopharmacological approaches provide clues for the search of bioactive compounds. Dryopteris ramosa (Hope) C. Chr. (plant family: Dryopteridaceae) is an ethnomedicinal plant of the Galliyat region of Pakistan. The aqueous fraction (AqF) of D. ramosa is being used by inhabitants of the Galliyat region of Pakistan to treat their gastrointestinal tract ailments, especially those caused by bacteria. The aims of the present study were as follows: (i) to justify the ethnomedicinal uses of the AqF of D. ramosa; (ii) to isolate a bioactive compound from the AqF of D. ramosa; and (iii) to evaluate the antibacterial and cytotoxic potential of the isolated compound. Column chromatography (CC) techniques were used for the isolation studies. Spectroscopic techniques (UV–Vis, MS, 1&2D NMR) were used for structural elucidation. The agar-well diffusion method was used to evaluate the antibacterial potential of “i3CβDGP” against five bacterial strains, and compare it with the known antibiotic “Cefixime”. The brine shrimp lethality test (BSLT) was used for cytotoxic studies. The AqF of D. ramosa afforded “iriflophenone-3-C-β-D glucopyranoside (i3CβDGP)” when subjected to LH20 Sephadex, followed by MPLC silica gel₆₀, and purified by preparative TLC. The “i3CβDGP” showed a strong potential (MIC = 31.1 ± 7.2, 62.5 ± 7.2, and 62.5 ± 7.2 µg/mL) against Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli, respectively. On the other hand, the least antibacterial potential was shown by “i3CβDGP” (MIC = 125 ± 7.2 µg/mL), against Bacillus subtilis, in comparison to Cefixime (MIC = 62.5 ± 7.2 µg/mL). The cytotoxicity of “i3CβDGP” was significantly low (LD₅₀ = 10.037 ± 2.8 µg/mL) against Artemia salina nauplii. This study not only justified the ethnomedicinal use of D. ramosa, but also highlighted the importance of ethnomedicinal knowledge. Further studies on AqF and other fractions of D. ramosa are in progress. Full article