Oxidative stress and neuroinflammation are crucial phenomena in the pathogenesis of Alzheimer disease (AD). Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons and modulates oxidative stress and neuroinflammation phenomena in rat brain. Based on this, our study investigates Oxo positive effects on differentiated SH-SY5Y neuroblastoma cells, revealing that Oxo treatment a) enhances cell survival, b) increases neurite length and c) upregulates HSP70 and pHSF1 expression. Moreover, Oxo counteracts tert–Butyl hydroperoxide (TBH)-induced cell death and reactive oxygen species (ROS) generation. The co-treatment with KNK437 (HSP70 inhibitor) reduces SOD1 expression and Oxo-induced protection against oxidative stress damage, suggesting the involvement of HSP70/SOD1 signaling in its effect. In addition, Oxo treatment enhances cell survival, increases neurite length and counteracts DNA fragmentation induced by Aβ1-42 peptide. The same treatment is also able to block ROS production and recover SOD impaired by Aβ1-42 peptide. Finally, Oxo recovers mitochondrial dysfunction associated with Aβ1-42, as evidenced by reduced mitochondrial superoxide levels, preserved mitochondrial membrane potential and mitigation of mitochondrial swelling. These results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD.