Preventing antifungal resistance development and identifying pathogens with high, medium, and low risk of resistance development to a particular fungicide or fungicide class is crucial in the fight against phytopathogens. We characterized the sensitivity of potato wilt-associatedFusarium oxysporumisolates to fludioxonil and penconazole and assessed the effect of these fungicides on the expression of fungal sterol-14-α-demethylase (CYP51a)and histidine kinase (HK1) genes. Penconazole stunted the growth ofF. oxysporumstrains at all concentrations used. While all isolates were susceptible to this fungicide, concentrations of up to 1.0 μg/mL were insufficient to cause a 50% inhibition. At low concentrations (0.63 and 1.25 μg/mL), fludioxonil stimulated growth inF. oxysporum. With an increase in the concentration of fludioxonil, only one strain (F. oxysporumS95) exhibited moderate sensitivity to the fungicide. Interaction ofF. oxysporumwith penconazole and fludioxonil leads to respective elevated expressions of theCYP51aandHK1genes, which upsurge with increasing concentration of the fungicides. The data obtained indicate that fludioxonil may no longer be suitable for potato protection and its continuous use could only lead to an increased resistance with time.