| Summary: | The ferns <i>Asplenium ceterach</i> L., <i>Asplenium scolopendrium</i> L. and <i>Asplenium trichomanes</i> L. have wide application in traditional medicine worldwide. However, the scientific research on their anticancer and antibacterial properties is insufficient. The present article aims to provide more information on this topic. Extracts derived from the aerial parts of <i>A. ceterach</i>, <i>A. scolopendrium</i> and <i>A. trichomanes</i> were examined using a panel of in vitro assays with different bacterial and mammalian cells. The cytotoxicity and anticancer activity of the samples were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays with three human (A549, FL, HeLa) and three murine (3T3, TIB-71, LS48) cell lines. Inhibitory effects on the growth of Gram-positive (<i>Bacillus cereus</i>) and Gram-negative (<i>Pseudomonas aeruginosa</i>) bacteria were determined by the agar diffusion assay. Apoptosis-inducing properties of the extracts were analyzed by flow cytometry. Superoxide dismutase (SOD) activity in extract-treated cells was investigated by ELISA. The obtained results demonstrate selective anticancer activity of all three <i>Asplenium</i> species. The extract from <i>A. ceterach</i> displayed the strongest inhibitory properties against human cervical cancer cells and bacterial cells. It induced a lower level of cytotoxicity against mouse cell lines, indicating a species-specific effect. The extract from <i>A. trichomanes</i> demonstrated better anticancer and antibacterial properties than the sample from <i>A. scolopendrium</i>. Further experiments linked the mechanism of action of <i>A. ceterach</i> extract with oxidative stress-inducing potential and strong proapoptotic potential against the cervical cancer cell line HeLa. <i>A. trichomanes</i> and <i>A. scolopendrium</i> extracts appeared to be potent inducers of necrotic cell death.
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