The aim of this study was to evaluate the biological and antimicrobial activities of commercial freeze-dried whey fermented by lactic acid bacteria in order to valorize this high polluting liquid waste of the dairy industry. Freeze-dried whey was fermented by different strains of Lactobacillus plantarum (CECT 220, 221, 748) at three different times of fermentation (24, 48, 72 h). Afterwards, the extract was purified on centricon amicon with a cut-off of 3 kDa to obtain a permeate consisting of small bioactive compounds reported in the literature to show greater bioactivity. The purified and diluted samples were subjected to the biological and antimicrobial tests for the evaluation of antioxidant, antihypertensive, iron binding, and antifungal activities and identification of phenolic compounds. The results highlighted a radical cation scavenging activity ranging from 1.415 to 2.083 mmol trolox equivalents TE per kg of dry weight, a percentage of iron binding capacity ranging between 23-55% and a percentage of ACE inhibitory activity ranging between 67-85%. The optimal biological activity was obtained from whey fermented by L. plantarum 220 for all the assays performed, except for the iron chelating activity. Furthermore, the antifungal analysis showed a good activity against the mycotoxigenic fungi belonging to Fusarium generum (F. moniliformis, F. graminearum and F. verticillioides), while a slight activity was obtained for Aspergillus and Penicillium generum. This antifungal activity could be correlated to the production of phenolic compounds during fermentation. The obtained results support the hypothesis of using whey as a functional ingredient to improve food preservation.