Achillea millefoliumL. is one of the most known medicinal plants with a broad spectrum of applications in the treatment of inflammation, pain, microbial infections and gastrointestinal disorders. In recent years, the extracts fromA. millefoliumhave also been applied in cosmetics with cleansing, moisturizing, shooting, conditioning and skin-lightening properties. The growing demand for naturally derived active substances, worsening environmental pollution and excessive use of natural resources are causing increased interest in the development of alternative methods for the production of plant-based ingredients. In vitro plant cultures are an eco-friendly tool for continuous production of desired plant metabolites, with increasing applicability in cosmetics and dietary supplements. The purpose of the study was to compare phytochemical composition and antioxidant and tyrosinase inhibitory properties of aqueous and hydroethanolic extracts fromA. millefoliumobtained from field conditions (AmL and AmH extracts) and in vitro cultures (AmIV extracts). In vitro microshoot cultures ofA. millefoliumwere obtained directly from seeds and harvested following 3 weeks of culture. Extracts prepared in water, 50% ethanol and 96% ethanol were compared for the total polyphenolic content, phytochemical content using the ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC-hr-qTOF/MS), antioxidant activity by DPPH scavenging assay and the influence on the activity of mushroom and murine tyrosinases. The phytochemical content of AmIV extracts was significantly different from AmL and AmH extracts. Most of the polyphenolic compounds identified in AmL and AmH extracts were present in AmIV extracts only in trace amounts and the major constituents presented in AmIV extracts were fatty acids. The total content of polyphenols in AmIV exceeded 0.25 mg GAE/g of dried extract, whereas AmL and AmH extracts contained from 0.46 ± 0.01 to 2.63 ± 0.11 mg GAE/g of dried extract, depending on the solvent used. The low content of polyphenols was most likely responsible for the low antioxidant activity of AmIV extracts (IC50values in DPPH scavenging assay >400 µg/mL) and the lack of tyrosinase inhibitory properties. AmIV extracts increased the activity of mushroom tyrosinase and tyrosinase present in B16F10 murine melanoma cells, whereas AmL and AmH extracts showed significant inhibitory potential. The presented data indicated that microshoot cultures ofA. millefoliumrequire further experimental research before they can be implemented as a valuable raw material for the cosmetics industry.