Recent studies have exhibited a very promising role of copper nanoparticles (CuNPs) in mitigation of abiotic stresses in plants. Arbuscular mycorrhizae fungi (AMF) assisted plants to trigger their defense mechanism against abiotic stresses. Arsenic (As) is a non-essential and injurious heavy-metal contaminant. Current research work was designed to elucidate role of CuNPs (100, 200 and 300 mM) and a commercial inoculum of Glomus species (Clonex® Root Maximizer) either alone or in combination (CuNPs + Clonex) on physiology, growth, and stress alleviation mechanisms of E. sibiricus growing in As spiked soils (0, 50, and 100 mg Kg− 1 soil). Arsenic induced oxidative stress, enhanced biosynthesis of hydrogen peroxide, lipid peroxidation and methylglyoxal (MG) in E. sibiricus. Moreover, As-phytotoxicity reduced photosynthetic activities and growth of plants. Results showed that individual and combined treatments, CuNPs (100 mM) as well as soil inoculation of AMF significantly enhanced root growth and shoot growth by declining As content in root tissues and shoot tissues in As polluted soils. E. sibiricus plants treated with CuNPs (100 mM) and/or AMF alleviated As induced phytotoxicity through upregulating the activity of antioxidative enzymes such as catalase (CAT) and superoxide dismutase (SOD) besides the biosynthesis of non-enzymatic antioxidants including phytochelatin (PC) and glutathione (GSH). In brief, supplementation of CuNPs (100 mM) alone or in combination with AMF reduced As uptake and alleviated the As-phytotoxicity in E. sibiricus by inducing stress tolerance mechanism resulting in the improvement of the plant growth parameters.