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Cytopiloyne (CP), a novel polyacetylene compound extracted from B. pilosa, shows a multi-bioactivity, including immunomodulatory and antidiabetes. Here, we investigated the anti-Listeria effect of cytopiloyne in mice by assessing mortality, clearance of L. monocytogenes, and pathology examination. The data presented herein are supplemental to our research article entitled “Cytopiloyne, a polyacetylenic glucoside from Bidens pilosa, acts as a novel anticandidial agent via regulation of macrophages” [1].