Nanotechnology is one of the most important recent trends to improve the production of secondary metabolites in tissue culture for wide commercial use. Therefore, in this study, the effects of magnetite nanoparticles (MNPs) of different sizes (10.77, 20.5, and 29.3 nm) and different concentrations (0.5, 1, and 2 ppm) on the growth and production of active constituents in Ginkgo biloba L callus were investigated. The best medium for callus growth and proliferation of Ginkgo biloba L was MS medium supplemented with 1 ppm naphthalene acetic acid (NAA), 1 ppm kinetin, and 0.5 ppm MNPs with a size of 10 nm, which gave the maximum fresh weight of callus and percentage increase in fresh weight after two subcultures. The maximum accumulation of ginkgolide A and bilobalide was obtained when the callus was stimulated by MNPs 10 nm at 2 ppm. This study also focused on the estimation of phenolic compounds by HPLC and free radical scavenging (antioxidant activity) with 2, 2-diphenyl-1-picrylhydrazyl (DPPH) for their indirect effect on the production of active constituents. In addition, MNPs of different sizes had a significant effect on the number of phenolic compounds, with maximum values recorded at (2 ppm + size 10.77 nm). The major components were quercetin, kaempferol, p-coumaric acid, rutin and Caffeic acid. Moreover, the highest percentage of antioxidant activity (DPPH) was recorded when the callus was treated with (2 ppm + size 10.77 nm), which reached 77.29%. This study highlights the importance of MNPs in the production of ginkgolides A and bilobalides from Ginkgo biloba L. and focuses on the relationship between their size and concentration. Moreover, to obtain a good and durable source of active compounds of medicinal and industrial importance at a large scale and at the lowest cost.