The chemical composition, cytotoxic effect, and antibacterial activity of protonema suspension cultures of Polytrichum juniperinum were evaluated. The most promising extracts from the field-collected moss were ethyl acetate and dichloromethane extracts with minimum inhibitory concentration (MIC) between 93.7 and 23.4 μg mL−1 in Bacillus cereus and Staphylococcus aureus, respectively, and dichloromethane extract from protonema suspension cultures with a MIC between 187.5 and 93.7 μg mL−1 in B. cereus. The extracts exhibited a dose-dependent cytotoxic effect against CHO-K1 cells. Ethyl acetate extract from field-collected moss (IC50: 61.2 μg mL−1) and dichloromethane extract from protonema suspension cultures (IC50: 65.6 μg mL−1) showed the greatest cell damage. Saponins, terpenoids, and flavonoids, protein (26%), Ca, K, and Mg were present in the tissue cultures.