Osmotic stress is one of the main destructive abiotic factors that hinder plant growth and development. In this research, the role of silver nanoparticles (Ag NPs) in mitigating the negative impact of osmotic stress on in vitro grown Chenopodium quinoa (Quinoa 6 Line; Q6) was investigated to determine whether Ag NPs were able to reduce the negative effects on the in vitro grown cultures of the Q6 line. The explants were subcultured onto a special osmostressing media containing sucrose, sorbitol, or mannitol at different levels (0.1, 0.2, 0.3, and 0.4 mol/L) to mimic the osmotic stressing environment for four weeks. Then, stress physiological responses of in vitro grown Q6 under the induced osmotic stress were investigated to determine the highest stress level that the microshoots could tolerate. Next, Ag NPs; 25, 50, and 75 mg/L were added to the medium that contained the highest stress level of the induced osmotic stress to determine if their addition improved the physiological performance of the Q6 microshoots under the most severe osmotic agent levels. The results revealed that 0.4 mol/L sucrose, 0.3 mol/L sorbitol, and 0.3 mol/L mannitol were the highest stress levels that the microshoots could tolerate. The addition of 75 mg/L Ag NPs to the previous highest stress levels resulted in a significant increase in the following: stem length (SL), leaves number (LN), fresh weight (FW), dry weight (DW), total chlorophyll, protein, calcium (Ca), and phosphorus (P) contents, while it caused a reduction in proline, sodium (Na) ions, and potassium (K) ions. These results indicate that the negative consequences of osmotic stress on Q6 quinoa microshoots could be mitigated by adding specific concentrations of Ag NPs to the culture medium.