Mucilage is a hydrophilic biopolymeric material of interest in the food industry due to its high content of dietary fiber, antioxidant activity, and gelling and thickening capacities, which is present in high concentration in agricultural by-products, such as the peel of cacti fruits. In this work, the powdered mucilage extracted from the peel of yellow pitahaya (Selenicereus megalanthus) fruit was characterized using a multi-technical approach that included proximal analysis (proteins, lipids, crude fiber, ash, and carbohydrates), as well as structural (FTIR, NMR, UPLC-QTOF-MS, and X-ray diffraction), colorimetric (CIELab parameters), morphological (SEM), and thermal (DSC/TGA) methods. Likewise, its total content of dietary fiber and polyphenols, as well as its antioxidant activity, were determined. This dried mucilage presented a light pale yellow-reddish color, attributed to the presence of betalains (bioactive pigments with high antioxidant activity). The FTIR spectrum revealed functional groups associated with a low presence of proteins (5.45 ± 0.04%) and a high concentration of oligosaccharides (55.26 ± 0.10%). A zeta potential of −29.90 ± 0.90 mV was determined, denoting an anionic nature that favors the use of this mucilage as a stable colloidal dispersion. UPLC-QTOF-MS analysis revealed a major oligosaccharide composition based on galacturonic acid units in anionic form. SEM micrographs revealed a cracked morphology composed of amorphous and irregular particles. According to the DSC/TGA results, this mucilage can be introduced as a new source of hydrocolloids in food processes since it has high thermal stability that has been manifested up to 373.87 °C. In addition, this biopolymer exhibited a high content of polyphenols (25.00 ± 0.01-g gallic acid equivalent (GAE)/100-g sample), dietary fiber (70.51%), and antioxidant activity (1.57 ± 0.01 mmol Trolox equivalents/kg of sample). It was concluded that this mucilaginous material presents sufficient physicochemical and functional conditions to be used as a nutritional ingredient, thus giving valorization to this agricultural by-product.