The fungicide difenoconazole, widely used to reduce the negative impacts of fungi diseases on areas with intensive farming, can reach freshwater systems causing deleterious effects on nontarget organisms. The acute and chronic toxicity of a commercial formulation containing 250 g L of difenoconazole (Prisma®) as the active ingredient was assessed in the freshwater planarian Girardia tigrina. The endpoints evaluated were feeding rate, locomotion, regeneration, and sexual reproduction of planarians. The estimated 48 h LC of the commercial formulation on planarians expressed as the concentration of the active ingredient difenoconazole was 47.5 mg a.i.L. A significant decrease of locomotion (LOEC = 18.56 mg a.i.L), delayed regeneration (LOEC = 9.28 mg a.i.L), and sexual reproduction impairment, i.e., decreased fecundity and fertility rates (LOEC ≤ 1.16 mg a.i.L) were observed on planarians exposed to sublethal concentrations of the formulation. This study demonstrated the importance of using reproductive, physiological, and behavioral parameters as more sensitive and complementary tools to assess the deleterious effects induced by a commercial formulation of difenoconazole on a nontarget freshwater organism. The added value and importance of our research work, namely, the impairment of sexual reproduction of planarians, contributes to the development of useful tools for ecotoxicology and highlights the fact that those tools should be developed as guidelines for testing of chemicals. Our results showed that the use of reproductive parameters of Girardia tigrina would help to complement and achieve a better assessment of the risk posed by triazole fungicides to freshwater ecosystems.