The prebiotic capacity of Pectin Oligosaccharides (POS) is influenced by structural factors such as molecular size, composition, and degree of esterification, which affect their interaction with the gut microbiota. While existing literature has predominantly examined POS derived from apple and citrus pectins, the extrapolation of these findings to other pectin sources remains complex due to variations in their composition. This study focused on obtaining POS with prebiotic potential from pisco grape pomace through controlled enzymatic hydrolysis, resulting in three molecular size fractions: <3 kDa, 3–10 kDa, and > 10 kDa. The POS fractions were analyzed using FTIR, HPSEC, HPLC, and MALDI-TOF-MS techniques to characterize their physical-chemical properties. Each fraction presented distinct compositions, with the <3 kDa fraction showing a higher concentration of galacturonic acid and glucose, while the >10 kDa fraction was also composed of rhamnose and arabinose. Notably, the <3 kDa fraction supported greater biomass growth of the probiotic strain Lactobacillus casei ATCC 393 compared to the other fractions. In contrast, the non-probiotic strain Escherichia coli ATCC 25922 achieved the lowest biomass with this fraction. Consequently, the <3 kDa POS fraction exhibited the highest prebiotic index. This fraction, composed of oligomers from the rhamnogalacturonan region and arabino-oligosaccharides with a degree of polymerization between two and five, highlights its potential for further research and applications. Therefore, investigating other sources and optimizing extraction conditions could lead to developing novel prebiotic formulations that supply specific probiotic strains for a symbiotic product.