Objective The microenvironment of breast epithelial tissue may contribute to the clinical expression of breast cancer. Breast epithelial tissue, whether healthy or tumoral, is directly in contact with fat cells, which in turn could influence tumor multifocality. In this pilot study we investigated whether the fatty acid composition of breast adipose tissue differed according to breast cancer focality. Methods Twenty-three consecutive women presenting with non-metastatic breast cancer underwent breast-imaging procedures including Magnetic Resonance Imaging prior to treatment. Breast adipose tissue specimens were collected during breast surgery. We established a biochemical profile of adipose tissue fatty acids by gas chromatography. We assessed whether there were differences according to breast cancer focality. Results We found that decreased levels in breast adipose tissue of docosahexaenoic and eicosa-pentaenoic acids, the two main polyunsaturated n-3 fatty acids of marine origin, were associated with multifocality. Discussion These differences in lipid content may contribute to mechanisms through which peritumoral adipose tissue fuels breast cancer multifocality.