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Lipolytic enzymes play a key role in the enzymatic degradation of whole wheat flour upon storage. These enzymes may also be involved in plant defense. Therefore, the objective of this study was to analyze differences in lipase (measured with olive oil or p-nitrophenyl palmitate as substrate), lipoxygenase, esterase (measured with p-nitropehnyl butyrate as substrate), and falling number (sprouting damage) in whole wheat flour from wheat that had been treated with and without foliar fungicide. Because different substrates can be used to quantify lipase activity, a secondary objective of this study was to determine if these substrates give similar results or not. Six hard red winter wheat genotypes grown in Nebraska with two treatments (foliar fungicide treated and untreated) for both (2014/15) years were analyzed. There was a significant treatment X year interaction for lipase, esterase, and lipoxygenase activities most likely because of high disease pressure in 2015. Fungicide treatment had a significant effect on falling number, lipase, and esterase activities in 2015 only. A significant genotype X treatment interaction was observed in lipoxygenase activity for both years. Positive Pearson partial correlation (year as a partial variable) was observed between lipase (measured with p-nitrophenyl palmitate) and esterase activities (r=0.63, p<0.001). Lipase activities measured with olive oil and p-nitrophenyl palmitate as the substrate were negatively correlated (r= -0.28, p<0.05). Falling number and lipoxygenase were not correlated to other variables. Results from this study suggested that enzymatic activities in the whole wheat flour is highly affected by the presence of disease pressure, and lipase activity measured with different substrates gave different responses.