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<i>Ageratina pichichensis</i>, is commonly used in traditional Mexican medicine. <i>In vitro</i> cultures were established from wild plant (WP) seeds, obtaining <i>in vitro</i> plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound’s identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0–2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in <i>in vitro</i> cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, <i>in vitro</i> cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, <i>A. pichichensis</i> WP and <i>in vitro</i> cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.