Growing interests in replacing conventional preservatives and antibiotics in food and pharmaceutical industries have driven the exploration of bacterial metabolites, especially those from strains with generally recognized as safe (GRAS) status, such as lactic acid bacteria (LAB). In this study, a supernatant cocktail derived from multiple LAB strains was prepared and its bioactivities-antimicrobial, antibiofilm, antioxidant, cytotoxicity, and stability-were thoroughly investigated. The cocktail's main components were identified using thermal and protease treatments, gas chromatography coupled to mass spectrometry (GC-MS), and flame ionization detection (GC-FID). The results demonstrated that the supernatant cocktail had a broad inhibition spectrum and was effective against food-related bacterial indicators with the highest activity observed on Bacillus cereus ATCC9634 (inhibition zone sizes 12.33 mm) and the lowest on Enterococcus faecium DSM 13590 (3.31 mm). It showed dose- and time-dependent delaying effects on the growth of tested fungi. Regarding the antibiofilm activity, it was more effective in preventing biofilm formation (40% biofilm mass reduction) than in degrading preformed biofilm (20% reduction). Additionally, the cocktail showed antioxidant capacity of 10.1 ± 0.3 g Trolox equivalent (TE)/kg and dose-dependent cytotoxicity on HEK-293 and HT-29 cell lines. The main bioactive compounds in this cocktail are organic acids (particularly acetic acid), volatiles, and bacteriocin-like compounds. The antimicrobial capacity of this supernatant cocktail was highly reproducible across different fermentation batches, and it remained highly stable at 4 °C. Overall, these findings provided novel insights into the functional potentials of LAB metabolites, broadening their application as customizable biopreservatives for food and pharmaceutical industry.