Difenoconazole is a triazole fungicide used to ensure sustainable agricultural, although it may also affect the aquatic environment. This study assessed the effects of this fungicide by both an experimental and a computational approach. The experimental assessment involved the acute exposure of Lemna minor to different concentrations of difenoconazole and the determination of main endpoints such as number of fronds and colony, as well as secondary endpoints represented by gravimetric, morphometrical and biochemical parameters. The in silico analysis consisted of the testing of difenoconazole and 14 of its transformation residues (TRs), using three computational tools (admetSAR, ADMETlab and T.E.S.T.) to assess either their toxicological endpoints (EC₅₀, IGC₅₀) or their probability of affecting a range of model aquatic organisms. The results highlighted a concentration-dependent effect of difenoconazole on both main and secondary endpoints. The calculated EC₅₀ value was 2.47 mg/L (data validated by EC₅₀ on Lemna gibba from Pesticides Properties DataBase), which categorizes difenoconazole as moderately toxic in the aquatic environment. The in silico assessment showed that two of the TRs showed lower toxicity, with these having only one aromatic ring compared to the others analyzed.