(1) Background: In this study, the potential use of Dioscorea rotundata hydrocolloids was evaluated to develop low-fat mayonnaise. (2) Methods: The effect of different concentrations of hydrocolloids on the physicochemical, microstructural, and rheological properties of mayonnaise was evaluated. (3) Results: Physicochemical analyses showed pH values that were stable over time but decreased with increasing hydrocolloid concentration. The color parameters showed a decrease in luminosity and an increase in the values of a* and b* over time, which can be translated into an increase in yellow and a decrease in white, with a greater accentuation in the control sample. The rheological study allowed us to obtain a non-Newtonian flow behavior of the shear-thinning type for all samples, and the flow curves were well-fitted by the Sisko model (R² ≥ 0.99). The samples had an elastic rather than viscous behavior, typical of dressings and emulsions. This indicates that the storage modulus was greater than the loss modulus (G′ > G″) in the evaluated frequency range. (4) Conclusions: hydrocolloids from Dioscorea rotundata have potential as a fat substitute in emulsion-type products.