Gluten-free products were initially for people with gluten-related disorders but are now popular with regular consumers. Research is investigating the health benefits of other gluten-free pseudo cereals, like quinoa, with higher nutritional value. This study aimed to explore the impact of substituting 30% rice flour (RF) in gluten-free mushroom instant dry soup with quinoa seed flour (QSF) at different levels on its nutritional, physicochemical, sensory characteristics, and the antioxidant activity. The results showed that replacing RF with QSF led to a significant improvement in the nutritional profile of the soup, with a linear increase in crude protein, fat, ash, crude fiber, total polyphenol, flavonoids, and antioxidant activity levels as the substitution rate increased. Moreover, macro- and microelements and essential amino acids increased significantly in the soup samples by adding QSF instead of RF, from 10 to 30%, compared to the control, which contained 30% of RF. However, the other total carbohydrate content decreased with the rise in the QSF concentration in the soup. In addition, substituting RF with QSF improved the soup samples’ rehydration ratio and total soluble solids. However, the color parameters (a* and b*) were increased with a decrease in L*. The sensory analysis revealed that the maximum substitution rate of QSF maintained the highest consumer acceptability, odor, flavor, texture, and appearance of the gluten-free mushroom instant soup samples. In conclusion, adding QSF to non-gluten soups improves their physical and chemical characteristics, nutritional impact, and organoleptic properties due to phytochemicals.