In this study, Limosilactobacillus fermentum PTCC 1638 and Lacticaseibacillus rhamnosus PTCC 1637 were used alone and in combination to ferment quinoa seeds, and the effect of fermentation (37 °C; 24 h) on the pH, total phenols, tocopherols, vitamin C, antioxidant activity, and enzymes inhibition (α-amylase and α-glucosidase; antidiabetic effect) was investigated. The results showed that with the increase in the fermentation time, the bacterial population, total phenols, antioxidant activity, and enzymes inhibition increased, which showed the greatest increase for the co-culture of L. rhamnosus and L. fermentum compared to the pure culture of each strain. Due to the increase in the fermentation time, the tocopherol isomers (α, β, γ, and δ), vitamin C, and pH decreased, and the largest decrease was related to the co-culture of the strains, followed by L. rhamnosus and L. fermentum. The results of this study showed that the co-culture and pure culture of bacteria can have different effects on the physicochemical properties and bioactive compounds of quinoa seeds.