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The total phenolic content (TPC), total tannin content (TTC) and total flavonoid content (TFC) as well as the antioxidant activity and the cytotoxic effect of the extract from leaves of Erythrochiton brasiliensis Nees & Mart. (Rutaceae) were evaluated. Raw material was collected in different European botanical gardens. Statistical analysis revealed a clear grouping of populations according to their climatic zone. The average TPC, TTC and TFC in tested samples were 35.92 (± 7.11) mg GAE·g–1 DW, 14.98 (± 4.08) mg PyE·g–1 DW and 2.92 (± 0.76) mg QuE·g–1 DW, respectively. The scavenged DPPH and Trolox equivalents determined by EPR spectroscopy were 1.23–4.14 and 0.50–1.44 mmol·g–1 of dry extract, respectively. Thirteen compounds (derivatives of bezoic acid acid and trans-cinnammic acid) were identified in the samples. The flavonoid vitexin was also present as the major component in three investigated samples. The in vitro cytotoxicity test of the extract on Vero cells provided IC50 and IC10 values of 175.6 and 72.5 μg·mL–1, respectively. Incubation of samples with HHV-1 infected Vero cells had no effect on the occurrence of cytopathic effect.