Background: This study aimed at exploring whether moderate ethanol drinking may have adverse effects on the fatty acids composition and on mitochondrial DNA (mtDNA) of rat brain. A secondary aim was to examine whether dietary antioxidant anthocyanins (ACN) can be protective. Methods: One group of rats received ethanol 12% and another water as an exclusive liquid to drink for 8 weeks. In order to test the impact of ACN consumption, two other groups of rats were fed an ACN-rich diet in combination with either ethanol or water. Brain fatty acids were measured by gas chromatography and mtDNA alterations, markers of mitochondrial suffering, were studied through an original real-time qPCR-based protocol. Results: Linoleic acid (LA, 18:2n-6) and eicosadienoic acid (20:2n-6) were significantly decreased, by 12% and 31% respectively, in the brains of both ethanol groups. The other brain lipids, including arachidonic acid (20:4n-6) and n-3 polyunsaturated fatty acids, were not modified. These changes were associated with a significant increase in deleted mtDNA (by 28%) in the ethanol group, without total mtDNA depletion. The ACN-rich diet prevented the increase in mtDNA common deletion (mtDNA CD). Conclusion: These data demonstrate that moderate ethanol drinking reduces certain brain n-6 and results in mtDNA injury. The antioxidant anthocyanins protect brain mtDNA but do not restore normal n-6 levels. Further studies are required to investigate the consequences of a decrease in n-6 levels in brain.