Eucryphia cordifolia Cav. native to the forests of southern Chile, was used for medicinal purposes by the Mapuche people in ancient times. However, there are no recent studies on the biological properties of its leaves in their natural habitat. This study assessed how the location of E. cordifolia populations in their ecological niche (center or periphery) impacts the chemical composition, antioxidant activity, and antimicrobial properties of leaf extracts.Ultra-Liquid Chromatography with Diode Array Detector, coupled to Quadrupole-Time of Fly Mass Spectrometry was used to characterize the chemical composition of the extracts (UHPLC-DAD-QTOF). The antioxidant activity was assessed through DPPH, ABTS, and ORAC assays, and the total polyphenol content was determined using the Folin-Ciocalteu method. Antimicrobial activity was analyzed using methods such as agar diffusion, determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and biofilm formation assay. The leaves extract contained 28 identified compounds, including flavonols, flavones, flavanols, and other bioactive compounds. The peripheral population exhibited higher antioxidant values based on ABTS and ORAC methods in autumn and spring, and higher total polyphenol levels only in autumn. The E. cordifolia extracts significantly inhibited the growth of E. coli, S. aureus, and P. aeruginosa, with minimum bactericidal concentration values ranging between 1.2 and 5.3 mg/mL, lower than in other studies. Additionally, the peripheral population displayed greater inhibition of C. glabrata biofilm formation compared to the centroid and intermediate populations in both summer and autumn. In conclusion, this first comprehensive investigation of E. cordifolia reveals that its leaves are a valuable source of bioactive compounds with biotechnological potential.