Milk fat is encapsulated in a milk fat globule membrane (MFGM) that contains bioactive glycoproteins and glycolipids. The MFGM inhibits infectivity of rotavirus (RV), activity that has been attributed to its glycoprotein and carbohydrate components. However, previous studies of proteins and oligosaccharides in the MGFM have not accounted for all the bioactivity associated with the complete MFGM. The lipid fraction of the MFGM accounts for half of its composition by weight, and we postulate that this fraction should be tested by itself to determine if it plays a role in antiviral activity. Herein, the anti-RV activity of an organic extract of MFGM was tested. Natural and whey buttermilk powders containing bovine MFGM enriched in polar lipids were prepared by microfiltration and supercritical fluid extraction treatment to reduce the triglyceride content of the powders. Lipid fractions were then extracted from the MFGM using both single- and dual-phase extraction methods. Whole MFGM and organic extracts were screened in MA-104 cells for anti-infective activity against a neuraminidase-sensitive rotavirus using a focus- forming unit assay. Dose-dependent inhibition was observed for whole buttermilk and cheese whey MFGM against the rotavirus. In general, buttermilk MFGM exhibited greater RV percentage inhibition than cheese whey MFGM. Organic-soluble anti-RV compounds were identified in bovine MFGM. The most active fraction, isolated by dual-phase extraction and iatrobead chromatography, was free of proteins and highly nonpolar. Further separation of this fraction in a less polar solvent (30:1 chloroform:methanol) resolved at least 5 lipid-containing compounds, which likely contribute to the anti-RV activity associated with bovine MFGM. In summary, lipid components associated with MFGM appear to contribute in large part to the anti-RV activity associated with the bovine MFGM.