Thioredoxin reductase 1 (TrxR1) has emerged as an important target for anticancer drug development due to its overexpression in many human tumors including breast cancer. Due to the serious side effects of currently used commercial anticancer drugs, new natural compounds with very few side effects and high efficacy are of great importance in cancer treatment. Lichen secondary metabolites, known as natural compounds, have diverse biological properties, including antioxidant and anticancer activities. Herein, we aimed to determine the potential antiproliferative, antimigratory, and apoptotic effects of evernic acid, a lichen secondary metabolite, on breast cancer MCF-7 and MDA-MB-453 cell lines and afterward to investigate whether its anticancer effect is exerted by TrxR1-targeting. The cytotoxicity results indicated that evernic acid suppressed the proliferation of MCF-7 and MDA-MB-453 cells in a dose-dependent manner and the IC50 values were calculated as 33.79 and 121.40 μg/mL, respectively. Migration assay results revealed the notable antimigratory ability of evernic acid against both cell types. The expression of apoptotic markers Bcl2 associated X, apoptosis regulator, Bcl2 apoptosis regulator, and tumor protein p53 by quantitative real-time polymerase chain reaction and western blot analysis showed that evernic acid did not induce apoptosis in both cell lines, consistent with flow cytometry results. Evernic acid showed its anticancer effect via inhibiting TrxR1 enzyme activity rather than mRNA and protein expression levels in both cell lines. In conclusion, these findings suggest that evernic acid has the potential to be evaluated as a therapeutic agent in breast cancer treatment.