Historically, botanical preparations have been used to improve human health. Their active ingredients are influenced by multiple factors as intraspecies variations, environmental conditions, collection time and methods and the part of the plant used. To ensure the efficiency and safety of these herbal drugs, qualitative and quantitative analyses are required. Tessaria absinthioides decoction (DETa) was reported as hypocholesterolemic, anti-inflammatory, cytotoxic, antitumor, and antioxidant. This work aimed to analyze DETa, by correlating its chemical composition with cytotoxic and antioxidant properties, to promote its research as an anticancer agent. DETa collections (2017, 2018, 2019 and 2022) were analyzed by UHPLC-UV-Vis, UHPLC-DAD-FLD and UPLC-MS/MS; cytotoxicity was assessed on the MCF-7 breast cancer cell line, and antioxidant capacity was evaluated by DPPH and FRAP methods; correlation analysis was used to determine biological and chemical markers. The results evidence that biological activities were consistent across collections. Among the quantified compounds, apigenin, naringin, gallocatechin gallate, ginnalin A, myricetin, epicatechin, OH-tyrosol, quercetin, and chlorogenic, tessaric, p-coumaric, vanillic, caffeic, caftaric, ellagic and rosmarinic acids correlated as bioactive and chemical markers. Moreover, tessaric acid could be established as a species marker. Altogether, these findings add relevant information to DETa properties encouraging further exploration of its potential application as anticancer botanical.