Phytochemical characterization and biological activities of many plants from Achillea genus have been described in scientific reports, while there are no published results regarding Achillea ageratifolia subsp. serbica (Nyman) Heimerl. Therefore, the aim of this paper was to investigate the different biological activities of methanolic extracts of this plant: antioxidant, antimicrobial, antibiofilm, genotoxic and antimutagenic, and also its phytochemical characterization. Results have demonstrated that extracts of different parts of the plant show significant antimicrobial activity against bacteria that cause food spoilage such as Staphylococcus aureus and Bacillus subtilis. This extracts also showed the highest antioxidant activity and significantly inhibits Pseudomonas aeruginosa ATCC 27853 to form the biofilm. HPLC analysis showed that flavonoid epicatechin was present in the root extract only, whereas rutin and quercetin were found in the extract of aerial part, while myricetin was only present in the inflorescence extract. The extracts of different parts of the plant were not genotoxic in all tested concentrations except the aerial part extract applied in the highest concentration, and also had a significant antimutagenic effect against known mutagens. Based on the results, methanolic extracts of the plant showed significant potential and these results could be used in the further investigation for the possible use in food protection and pharmacy.