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A nutritional approach could be a promising strategy to prevent or decrease the progression of neurodegenerative disorders such as Parkinson’s disease (PD). The neuroprotective role of walnut oil (WO) was investigated in <i>Drosophila melanogaster</i> treated with rotenone (Rot), as a PD model, WO, or their combination, and compared to controls. WO reduced mortality and improved locomotor activity impairment after 3 and 7 days, induced by Rot. LC-MS analyses of fatty acid levels in <i>Drosophila</i> heads showed a significant increase in linolenic (ALA) and linoleic acid (LA) both in flies fed with the WO-enriched diet and in those treated with the association of WO with Rot. Flies supplemented with the WO diet showed an increase in brain dopamine (DA) level, while Rot treatment significantly depleted dopamine content; conversely, the association of Rot with WO did not modify DA content compared to controls. The greater intake of ALA and LA in the enriched diet enhanced their levels in <i>Drosophila</i> brain, suggesting a neuroprotective role of polyunsaturated fatty acids against Rot-induced neurotoxicity. The involvement of the dopaminergic system in the improvement of behavioral and biochemical parameters in <i>Drosophila</i> fed with WO is also suggested.