Background: Spent coffee grounds (SCG) are a promising source of natural by-products which can be used for different purposes. In this work, a possible use of oligosaccharides isolated from SCG as functional ingredients was investigated. SCGs were treated with an acid hydrolysis at high temperature (200 °C) in a closed reactor setting reaction time of 30, 60 and 90 s depending on the sample (original or defatted). A comprehensive study of the resulted water-soluble hydrolysate using a high-resolution mass spectrometry analysis was performed. Additionally, the growth of four Lactobacillus strains was tested to assess the prebiotic potential of the hydrolysate. Results: Oligosaccharide chains formed by hexoses with a degree of polymerization ranging from 3 to 6 were identified and characterized. Regardless of the composition and the reaction time of hydrolysis, the bacterial activity of SCG extracts exhibited significantly higher values than the well-known versatile carbohydrate used by food industry, i.e., inulin. Conclusions: The results pave the way toward the use of hydrolysate SCG as an innovative ingredient intended to fortify food formulations. The diversity in coffee oligosaccharides composition suggests their selective prebiotic activity for specific bacterial strains. Graphical Abstract: [Figure not available: see fulltext.]