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In recent decades, natural products of plant origin have predominated among the sources of new therapeutic agents. Plants, which are intensively used in folk medicine, are the focus of particular interest, and being subjected to in-depth analyses for the assessment of their pharmacological properties. The objective of the present work was to evaluate the in vitro anticancer, antiviral, and antioxidant properties of a crude aqueous methanolic extract from the leaves of Rhus typhina L., which is a plant species with broad applications in traditional phytotherapy. The anticancer capacity of the extract was assessed on two human breast cancer cell lines (MCF7 and MDA-MB-231) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay, and its cell growth inhibitory effect on cancer cells was compared with its action on a non-cancerous breast epithelial cell line (MCF10A). Moreover, the in vitro antiviral activity of the plant extract was studied against viral strains of three taxonomic groups: herpes simplex virus type 1 (HSV-1), coxsackievirus B1 (CVB1), and influenza A virus (IAV/H3N2) using a cytopathic effect inhibition test, extract cytotoxicity evaluation through the neutral red uptake assay, a virucidal assay, a viral attachment assay, and cell pre-treatment with the extract. In turn, a radical scavenging assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH) was applied to explore the antioxidant potential of R. typhina leaf extract. Results indicated that the plant extract possessed antiproliferative activity against the tested cell lines which was stronger toward cancer cells. A modest inhibitory effect on IAV/H3N2 replication was detected; as well as on an effect on herpes virions and on the adsorption of the HSV-1 and IAV/H3N2 strains; and a protective effect on uninfected cells before HSV-1 contact. The leaf extract of R. typhina also exhibited a strong free radical scavenging activity. The results of the present work demonstrated the pharmacological potentials of the plant extract, which warrants further and more detailed study in the future. [ABSTRACT FROM AUTHOR]