Neuroinflammation, depletion of acetylcholine and neuronal oxidative stress are hallmarks of neurodegenerative diseases such as Alzheimer’s disease. This study sought to investigate in vitro acetylcholinesterase inhibitory activity, free radical scavenging, antioxidant activity, and anti-inflammatory activity of aqueous leaf extract of Juglans regia. The antioxidant, anti-inflammatory, and acetylcholinesterase-inhibitory activities of the extract were evaluated using different appropriate techniques. The extract showed substantial free radical scavenging and antioxidant activity by markedly scavenging hydroxyl radical, DPPH, nitric oxide, hydrogen peroxide, with concomitant high free radical reducing power. Similarly, lipid peroxidation and inflammation were significantly inhibited by the extract. The extract elicited acetylcholinesterase-inhibitory potential of 45.49%, suggesting they could be beneficial in managing neurodegenerative diseases. These biological activities can be attributed to the phytochemical constituents of the extract. Preliminary phytochemical screening of the extract showed the presence of polyphenols, flavonoids, and tannins and GCMS identified phytochemicals including vanillic acid, p-Coumaric acid, caffeic acid, squalene. This study concluded that the aqueous leaf extract of J. regia exhibited significant antioxidant, anti-inflammatory and acetylcholinesterase-inhibitory activities which could be beneficial in mitigating the pathological features of Alzheimer’s disease and other neurodegenerative diseases, thus, it could be explored as health-promoting dietary supplements/ products.