2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic herbicide. It provokes lipid peroxidation (LPO), oxidative stress, inflammation, and apoptosis in mammalian cells. The purpose of this study is to investigate the regulatory effects of flaxseed oil (FSO), and fish oil (FO) against the combined toxicity of 2,4-D and gamma-radiation (2,4-D/R) in rats' liver, kidney, testis, and brain tissues. 2,4-D/R treated rats demonstrated significant alterations in the activity of the hepatic enzymes (ALT, AST, ALP, and LDH), the levels of urea, creatinine (Creat), total cholesterol (TC), triglycerides (TG), total protein (TP), albumin (ALB), reproductive hormones (Testosterone (TH), Follicle Stimulating Hormone (FSH), and Luteinizing Hormone (LH)), cytokines (IL-1β, IL-6, TNF-α, NF-κB), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes in serum. In addition, 2,4-D/R intoxicated rats showed significant changes in the levels of malondialdehyde (MDA), glutathione reduced (GSH), and trace elements (Ca2+, Fe3+, Cu2+, and Zn2+), as well as significant alterations in the gene expression ratios of the apoptotic markers (BAX, BCL2, cytochrome c, caspases-9, and caspase-3) in the liver, kidney, testis, and brain tissues. Moreover, 2,4-D/R demonstrated lower sperm count and motility. FSO and FO-treated 2,4-D/R groups exhibited significant amelioration in the changes in the investigated parameters. The histopathological investigation confirmed the biochemical investigations. In conclusion, FSO and FO showed regulatory effects on 2,4-D/R toxicity-induced damage to liver, kidney, testis, and brain, via regulation of the mitochondrial apoptotic pathway and trace elements' levels.