Chemical investigation of the dichloromethane extract of Gundelia tournefortii L. afforded mevalonolactone by gas chromatography-tandem mass spectrometry. The mevalonolactone fraction was further studied for quorum quenching against enteropathogenic Escherichia coli (EPEC) infection and virulence on human enterocytes (CRL-1831) including cytoprotective and immunomodulatory activities. Significant downregulation of expressed EPEC virulence genes, eaeAg (intimin), escC (type III secretion biogenesis), and tir (translocated intimin receptor), was observed by qRT-PCR compared to controls (P < 0.05). The fraction did not exhibit cytoxicity in CRL-1831 (IC50 >100 µg/mL). EPEC infection challenge of CRL-1831 previously treated with the non-toxic fraction demonstrated inhibition of pathogen-induced apoptosis by downregulated expression of the early apoptosis marker cjun, accompanied by upregulated expression of the anti-apoptotic factor A20. Expression of huGal1-R in EPEC-infected set-ups was not affected suggesting non-disruption of facilitated bacterial clearance. Moreover, the fraction also showed anti-inflammatory activity by significantly downregulating the expression of pro-inflammatory IL-8 and TNF-α cytokine genes compared to controls (P < 0.05). Simultaneously, fluorescence photomicroscopy revealed cytoprotective activity against EPEC pathogenesis by blockade of intimin-mediated host cell attachment. LigandFit molecular docking of mevalonolactone showed binding in the EPEC TIR intimin binding region inferring the reason behind the impedance of attachment on CRL-1831 cells. These findings support evidence on the anti-pathogenic, anti-inflammatory and chemoprotective activities of mevalonolactone from G. tournefortii L. during host-pathogen interaction. To the best of our knowledge, this is the first report on the isolation of mevalonolactone from G. tournefortii L.