Bioprospecting is the identification and exploitation of natural resources for commercial purposes. Cheese whey is a major dairy by-product rich in nutrients and a possible source of bioactive compounds. The aim of this study was to isolate and identify lactic acid bacteria from cheese whey and characterize them based on their probiotic and technological properties, together with their cytotoxicity and cytoprotective effect on intestinal epithelial cells. From 142 isolates, three were selected as potential probiotic strains; all three were identified as Limosilactobacillus fermentum by 16S rRNA gene sequencing. The strains co-aggregated with pathogenic bacteria (up to 52.7%), scavenged the ABTS^●+ radical cation (10–70%), produced 374–459 mg/L of exopolysaccharides and tolerated sucrose, NaCl and CaCl₂ at the concentrations tested. L. fermentum VM016, VM023 and VM030 showed no cytotoxicity at 10⁷ CFU/mL and showed a cytoprotective effect against the oxidative stress of menadione at concentrations as low as 10⁴ CFU/mL in IEC-18 cells, and 10⁵ CFU/mL in Caco-2 cells. These results show that cheese whey retains a cultivable lactic acid bacteria fraction already adapted to the physicochemical conditions of the dairy matrix, from which autochthonous cultures can be recovered exhibiting probiotic potential, technological suitability, and favorable preliminary safety indicators for human consumption.