Isatis spp. are well-known for their industrial significance due to natural sources of indigotin and indirubin, important indole alkaloids, used in the dye and pharmaceutical industries. In this study, silver nanoparticles (AgNP) and salicylic acid-chitosan nanoparticles (SA-CNP) were synthesized and applied to enhance the production of indigotin and indirubin in shoot and root cultures of Isatis tinctoria and Isatis ermenekensis. Different doses of AgNP and SA-CNP were administered to three-week-old shoot and root cultures, and the effects were assessed at 12, 24, and 48 h. The harvested samples were analyzed to quantify indigotin and indirubin levels. Furthermore, the expression levels of It-TSA and CYP79B2 genes, known to be involved in indole alkaloid biosynthesis, were determined. In I. tinctoria roots, the highest levels of indigotin and indirubin were observed after applying 150 mg L−1 of SA-CNP for 48 h while in I. ermenekensis shoots, indigotin and indirubin reached the maximum levels with the application of 8 mg L−1 AgNP for 48 h. NP application had no remarkable effects on the accumulation of indigotin and indirubin in I. tinctoria shoots and I. ermenekensis roots compared to controls. Additionally, shoot cultures demonstrated superior indirubin production, which significantly increased with AgNP applications. The gene expression analysis also exhibited significant correlations with the changes in indigotin and indirubin levels. The findings of this study lay the groundwork for enhancing in vitro production of indigotin and indirubin in Isatis species through NP applications, and for developing high-capacity production strategies by determining optimal dosages in scale-up studies.