Nowadays, a strong ascending interest is given to the use of nanoparticles in soils to evaluate their benefits or risks to plants and microbes. Pea (Pisum sativum L.) of the family Fabaceae is among the crops cultivated by farmers in the winter season. From here, this study was conducted to examine the impact of arbuscular mycorrhizal fungus (AMF) and zinc ferrite nanoparticles (nano-ZnFe2O4) co-application on biochemical parameters and mineral contents of pea plants, as well as the impact of nano-ZnFe2O4 application on mycorrhizal root colonization. For this purpose, nano-ZnFe2O4 with a crystallite size of 25.4 nm was used in two different concentrations (0 and 5 µM) in pots planted with pea plants and inoculated or non-inoculated with AMF. The results showed that nano-ZnFe2O4 to some extent inhibited AMF colonization of pea plant roots. Additionally, in comparison to non-AM controls, the combined use of AMF and nano-ZnFe2O4 was successful in raising the activity of helpful enzymes (acid phosphatase, alkaline phosphatase, catalase and peroxidase) as well as biochemical parameters including primary metabolites (carbohydrates, protein, and amino acids). Moreover, an improvement in mineral nutrients uptakes in pea plants such as N, P, K and Mg appeared in the combined treatment. Therefore, nano-ZnFe2O4 coupled with AMF promises potential applications in agriculture because Zn and Fe are two essential micronutrients that need to be supplied to crop plants.