Background: Few studies have implemented biomarkers of fatty acid intake in relation to breast cancer. Aims: To examine possible differences in adipose tissue fatty acid composition between breast cancer patients and healthy control women. The relationship between tumor promotion and adipose tissue fatty acid synthesis was also investigated. Methods: The study was conducted at the University of Crete. Subjects included 94 women with clinically diagnosed cancer of the breast and 131 healthy control women. Histological tumor grading and breast cancer staging were assessed. Fatty acids were determined by gas chromatography in gluteal adipose tissue. Results: Conditional logistic regression analysis controlling for potential confounders indicated that elevated adipose monounsaturated fatty acids and oleic acid are associated with reduced odds of breast cancer [OR (T2 vs. T1) 0.15; 95% CI 0.03–0.64, and OR (T2 vs. T1) 0.18; 95% CI 0.04–0.71, respectively]. Adipose myristic acid was associated with an increase in breast cancer risk [OR (T3 vs. T1) 5.66; 95% CI 1.3–23.9]. Conclusions: Adipose oleic acid is inversely related, whereas adipose myristic acid is positively related to breast cancer risk. These relations could be mediated by Her-2/neu and FAS oncogenes.