In the recent years, 4-methylumbelliferone (4-MU) has been gaining importance, both as an anti-cancer agent and as a dietary supplement. The aim of this study was to determine the effectiveness of 4-MU as a carbon source for potential probiotic bacteria Lactobacillus helveticus 2126. For this purpose, a series of plate assays and infrared spectroscopy (FTIR) were used for 4-MU before and after the treatment with L. helveticus 2126. The plate assays indicated an initial inhibition followed by utilization of 4-MU that stimulated bacterial growth. A significant shift was observed in the FTIR peaks, which also have suggested possible extracellular activity of the bacteria for 4-MU utilization.