Background: Microalgae and nanoparticles are currently considered promising tools for numerous agricultural and biotechnological applications. The green microalga Chlorellasp. MF1 and its biosynthesized silver nanoparticles (AgNPs) were used in this study as biofortification agents to enhance glucosinolate and kaempferol levels in Eruca sativa. UV–visible spectroscopy, XRD, FTIR and TEM were comprehensively used for characterizing Chlorella-based AgNPs. Results: The biosynthesized AgNPs were found to be spherical in shape, with size ranging from 1.45 to 5.08 nm. According to FTIR measurements, silver ions were reduced to AgNPs by functional groups such as amide, hydroxyl and carboxylate. Different experimental treatments were conducted, including either soaking seeds of E. sativaor foliar spray with various concentrations of Chlorellasuspension (1, 2, 3 and 4 g L−1) and AgNPs (5, 10, 20 and 40 mg L−1). Expression levels of five key genes in the biosynthetic pathway of glucosinolates (MAM1, SUR1, MYB34and MYB51) and kaempferol (CHS) were assessed using qRT-PCR. The results indicated an upregulation in the gene expression levels in all treatments compared to control, recording the highest level at 40 mg L−1AgNPs and 4 g L−1Chlorellasuspension. In addition, high glucosinolates and kaempferol content was detected in plants whose leaves were sprayed with AgNPs and Chlorellasuspension (40 mg L−1and 4 g L−1) based on HPLC analysis. Sequence analysis of amplified CHSfragments from E. sativaplants treated with AgNPs (40 mg L−1) showed high sequence similarity to A. thaliana CHSgene. However, there were several CHSregions with sequence polymorphism (SNPs and Indels) in foliar sprayed plants. Conclusions: Results of this study evidenced that the application of AgNPs and Chlorellasuspension increased glucosinolates and kaempferol content in E. sativathrough upregulation of key genes in their biosynthetic pathway.