This study investigated the effects of magnetite nanoparticles (MNPs) various 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. 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 were achieved when callus was elicited by MNPs 10 nm at 2 ppm. This study also focused on estimating phenolic compounds and scavenging free radicals (antioxidant activity) for their indirect effect on active constituents production. Also, MNPs different sizes had a clear effect on the number of phenolic compounds, where the maximum values were recorded with (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 callus treated with (2 ppm + size 10.77 nm) which reached 77.29 %. This study highlights the importance of MNPs for the production of ginkgolide A and bilobalide from Ginkgo biloba L. and focusing on the relationship between their size and concentration. Besides, to provide a good and permanent source of active substances of medical and industrial importance on a large scale and at the lowest cost.