The use of essential oils (EOs) is known since long time in traditional medicine and aromatherapy for the management of various oxidative stress-related disorders and has been further increased recently for their neuroprotective and anti-aging potentials as well as for reducing anxiety and stress. The purpose of this work was to evaluate, for the first time, the chemical composition of Citrus lumia Risso EO and its antioxidant, anti-cholinesterase, and neuroactive properties by cell-free and cell-based assays. The EO has shown strong antioxidant and free radical scavenging properties, particularly in hydrogen atom transfer based assays (β-carotene bleaching and ORAC, IC 22 μg/mL and 46 μg/mL, respectively), that can be attributed to the high content of monoterpenes, especially D-Limonene (48.905%), and Linalool (18.245%). Furthermore, the EO has shown an interesting anti-acetylcholinesterase activity (IC 258.25 μg/mL). Data from MTT analysis indicate that the cytotoxicity of EO, evaluated on L929 mouse fibroblasts, is very low, with an IC higher than 500 μg/mL at 48 h. Rat neuronal networks subjected to EO showed a concentration-dependent inhibition of spontaneous electrical activity. Results indicate that C. lumia EO could be an important source of natural antioxidants suggesting an important preventive role in the onset of oxidative stress-related pathologies.