As rhizobacteria have extensively been studied for the production of compounds with biocontrol properties, methods to enhance its production are very important. In this study, nanotechnological method to modulate the rhamnolipid production by Pseudomonas sp. K6 has been demonstrated. For this, Pseudomonas sp. K6 was cultured with different concentrations of chitosan nanoparticles (CNPs) and gold nanoparticles (AuNPs). The rhamnolipid production was further analyzed by CTAB- methylene blue agar assay and also by LC–MS/MS analysis. From the LC–MS/MS result, induction of dirhamnolipid production in K6 could be confirmed when cultured with CNPs (1, 5 and 10 mg/mL) and AuNPs (10, 25, 50 and 100 µg/mL). The monorhamnolipid production by K6 was observed to get enhanced when the K6 culture was supplemented with 2.5 mg/mL CNPs and 10 µg/mL AuNPs. Also, in planta study confirmed the biocontrol ability of rhamnolipid as it suppressed the Sclerotium rolfsii infection in Vigna unguiculata plants. As the rhamnolipids have versatile applications in the agricultural field, the nano-based approach to enhance its production from the biocontrol organism is significant.