Alzheimer’s disease (AD) is a prevalent neurodegenerative disorder characterized by significant cognitive impairment and predominantly affects the elderly. With no effective cure available, research continues to explore novel therapeutic and preventive strategies. Palmitic acid-9-hydroxystearic acid (9-PAHSA), a novel class of bioactive lipids with anti-inflammatory and anti-diabetic properties, has shown potential as a dietary supplement. Mitochondrial dysfunction is recognized as a significant pathological feature of AD, with impaired mitophagy leading to the accumulation of dysfunctional mitochondria, thus exacerbating disease progression. This study evaluates the hypothesis that S-9-PAHSA can ameliorate cognitive dysfunction in AD by enhancing mitochondrial autophagy in 5xFAD mice. The treatment group received S-9-PAHSA in their drinking water for three months. Behavioral studies were conducted using the Morris Water Maze (MWM) and Y-Maze, with further assessments of amyloid-beta (Aβ) plaque deposition, neuronal apoptosis, and mitochondrial health. S-9-PAHSA significantly enhanced spatial learning and memory, reduced amyloid plaque deposition, decreased neuronal apoptosis, and improved mitochondrial health and autophagy in 5xFAD mice. These findings suggest that S-9-PAHSA holds promise as a supplementary preventive and therapeutic strategy for Alzheimer’s disease treatment.