Perilla seed oil, derived from a regional plant native to northern Thailand, undergoes cold‐pressing to analyze its bioactive components, notably alpha‐linolenic acid (ALA). ALA, constituting approximately 61% of the oil, serves as a precursor for therapeutic omega‐3 fatty acids, EPA and DHA, with neurodegenerative disease benefits and anti‐inflammatory responses. This study administered different concentrations of perilla seed oil to male C57BL/6 mice, categorized as low dose (LP 5% w/w), middle dose (MP 10% w/w), and high dose (HP 20% w/w), along with a fish oil (FP 10% w/w) diet. An experimental group received soybean oil (5% w/w). Over 42 days, these diets were administered while inducing Parkinson's disease (PD) with rotenone injections. Mice on a high perilla seed oil dose exhibited decreased Cox‐2 expression in the colon, suppressed Iba‐1 microglia activation, reduced alpha‐synuclein accumulation in the colon and hippocampus, prevented dopaminergic cell death in the substantia nigra, and improved motor and non‐motor symptoms. Mice on a middle dose showed maintenance of diverse gut microbiota, with an increased abundance of short‐chain fatty acid (SCFA)‐producing bacteria (Bifidobacteria, Lactobacillus, and Faecalibacteria). A reduction in bacteria correlated with PD (Turicibacter, Ruminococcus, and Akkermansia) was observed. Results suggest the potential therapeutic efficacy of high perilla seed oil doses in mitigating both intestinal and neurological aspects linked to the gut–brain axis in PD.