Helichrysum plicatum subsp. polyphyllum (H. plicatum), is an important species belonging to the family Acteraceae, which is used in alternative medicine. For this reason, it was aimed to determine the antimicrobial and cytotoxic activity of H. plicatum extract in this study. Firstly, H. plicatum was extracted with methanol for 6 hours using a Soxhlet. Secondly, the antimicrobial activity of the extract was tested against Gram-positive (Staphylococcus aureus ATCC®25923, Bacillus cereus ATCC®7064, Bacillus subtilis ATCC®6633) and Gram-negative (Escherichia coli ATCC®25922, Klebsiella pneumoniae ATCC®706003, Salmonella Enteritidis ATCC®13076) bacteria and a yeast (Candida albicans ATCC®10231) using well diffusion and microdilution methods. Finally, the cytotoxic activity of H. plicatum extract on DLD-1 (human colon adenocarcinoma) and CCD-18Co (normal colon) cell lines was determined by MTT method. According to the results of the antimicrobial effect, H. plicatum extract was found to have a stronger antibacterial activity against Salmonella Enteritidis (24.13±1.15 and 156µg/mL) among Gram-negative bacteria. It was determined to have inhibitory activity against also Bacillus cereus (16.66±1.52 and 312µg/mL) among Gram-positive bacteria. In addition, the extract showed a strong antifungal effect on Candida albicans (22.66±0.57 and 312µg/mL). In addition, the H. plicatum extract did not show any cytotoxic effect on CCD-18Co cells, while the IC50 value on the DLD-1 cell line was determined as 8.17mg/mL. This indicates that the plant extract has a selective toxic effect on cancer cells. These results reveal that H. plicatum has the potential to be used for therapeutic purposes after being supported by further studies.