Paraquat (PQ) (1,1'-dimethyl-4-4'-bipyridinium dichloride) is the second most widely used herbicide worldwide; however, in countries different sales and distribution remain restricted. Chronic exposure to PQ leads to several diseases related to oxidative stress and mitochondrial dysfunctions including myocardial failure, cancer, and neurodegeneration and subsequently death depending upon the dose level. The aim of this study was to examine if diet supplementation with eicosapentaenoic and docosahexaenoic acids (EPA and DHA, omega-3 long-chain fatty acids) serves a protective mechanism against neuromuscular dysfunctions mediated by PQ using Drosophila melanogaster as a model with focus on mitochondrial metabolism. PQ ingestion (170 mg/kg b.w. for 3 d) resulted in a decreased life span and climbing ability in D. melanogaster. In the brain, PQ increased thioflavin fluorescence and reduced either 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclei staining and neuronal nuclei protein (NeuN) positive neurons, indicating amyloid formation and neurodegenetation, respectively. In the thorax, PQ ingestion lowered citrate synthase activity and respiratory functions indicating a reduction in mitochondrial content. PQ elevated Ca2+/calmodulin-dependent protein kinase II (CaMKII) mRNA expression levels, indicative of high calcium influx from cytosol to mitochondrial matrix. In brain and thorax, PQ also increased hydrogen peroxide (H2O2) production and impaired acetylcholinesterase (AChE) activity. Concomitant EPA/DHA ingestion (0.31/0.19 mg/kg b.w.) protected D. melanogaster against PQ-induced toxicity preserving neuromuscular function and slowing down the rate of aging. In brain and thorax, these omega-3 fatty acids inhibited excess H2O2 production and restored AChE activity. EPA/DHA delayed amyloid deposition in the brain, and restored low citrate synthase activity and respiratory functions in the thorax. The effects in the thorax were attributed to stimulated mRNA expression level of genes involved either in mitochondrial dynamics or biogenesis promoted by EPA/DHA: dynamin-related protein (DRP1), mitochondrial assembly regulatory factor (MARF), mitochondrial dynamin like GTPase (OPA1), and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). In conclusion, diet supplementation with EPA/DHA appears to protect D. melanogaster muscular and neuronal tissues against PQ intoxication.