The impacts of water deficit and melamine salt of bis(oximethyl)phosphonic acid (melaphen) on the fatty acid (FA) composition of membrane lipids and energy metabolism in mitochondria of 5-day-old pea (Pisum sativum L., cv. Flora-2) seedlings were studied. Insufficient watering resulted in the accumulation of saturated and a decrease in the content of unsaturated FAs with 18 and 20 carbon atoms. Seed treatment with 3 × 10−10 M melaphen prevented these changes in the FA composition in the mitochondrial membrane lipids. Changes in the FA compositions of membrane lipids were correlated with changes in energy metabolism in mitochondria: the efficiency of oxidative phosphorylation and the rate of NAD-dependent substrate oxidation in the presence of ADP and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) were reduced. A close correlation was observed between changes in the highest rates of NAD-dependent substrate oxidation and the relative content of FAs with 18 (r = 0.76489) and 20 (r = 0.9637) carbon atoms. The regulatory role of C18 and C20 unsaturated FAs in the mitochondrial energy metabolism of pea seedlings is discussed.