This study investigated the therapeutic and preventive potential of the ethanolic extract of Withania somnifera [Ashwagandha] and its isolated fatty acid, linoleic acid, against trichloroethylene [TCE]-induced neurodegenerative disorders in male Wistar rats. A total of eight experimental groups were established: a negative control (distilled water), a positive control [TCE], and six treatment groups receiving linoleic acid, palmitic acid, the crude ethanolic extract, or combinations thereof, either prophylactically or therapeutically. Biochemical assessments were performed to determine serum and brain levels of acetylcholine, vitamin B12, and linoleic acid. Rats exposed to TCE exhibited significant reductions in vitamin B12, palmitic acid, and linoleic acid levels, alongside elevated acetylcholine compared with the negative control. In contrast, treatment with linoleic acid, palmitic acid, the ethanolic extract, or their combinations significantly restored vitamin B12 and linoleic acid levels while reducing acetylcholine concentrations. These findings indicate that Ashwagandha extract and linoleic acid exert both therapeutic and protective effects against TCE-induced neurotoxicity by improving biochemical parameters, highlighting their potential as promising candidates for managing neurodegenerative disorders.