The present study investigates the impact of varying concentrations of PVC microplastics (PVC–MPs) – specifically 0 (no PVC–MPs), 2, and 4 mg L− 1 –alongside different arsenic (As) levels of 0 (no As), 150, and 300 mg kg− 1 in the soil, with the concurrent application of copper oxide–nanoparticles (CuO–NPs) at 0 (no CuO –NPs), 25 and 50 µg mL− 1 to barley (Hordeum vulgare L.) plants. This research primarily aims to assess plant growth and biomass, photosynthetic pigments and gas exchange characteristics, oxidative stress indicators, as well as the response of various antioxidants (both enzymatic and non-enzymatic) and their relevant genes expression, proline metabolism, the AsA–GSH cycle, and cellular fractionation within the plants. The findings showed that increased levels of PVC–MPs and As stress in the soil significantly reduced plant growth and biomass, photosynthetic pigments, and gas exchange characteristics. Additionally, PVC–MPs and As stress increased oxidative stress in the roots and shoots, as evidenced by elevated levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), and electrolyte leakage (EL), which in turn stimulated the production of various enzymatic and non-enzymatic antioxidants, gene expression, and sugar content. Furthermore, a notable increase in proline metabolism, the AsA–GSH cycle, and cellular pigmentation was observed. Conversely, the application of CuO–NPs resulted in a substantial improvement in plant growth and biomass, gas exchange characteristics, and the activity of enzymatic and non-enzymatic antioxidants, along with a reduction in oxidative stress. Additionally, CuO–NPs enhanced cellular fractionation while decreasing proline metabolism and the AsA-GSH cycle in H. vulgare plants. These outcomes provide new insights into sustainable agricultural practices and offer significant potential in addressing the critical challenges of heavy metal contamination in agricultural soils.