Bacteriocins from LAB that have a wide antimicrobial spectrum against Gram-negative and Gram-positive bacteria and fungi have not frequently been observed. Many bacteriocins could not inhibit Gram-negative bacteria (Gao, Jia, Gao, & Tan, 2010). Salivaricin SMXD51 (Messaoudi et al., 2012) inhibited the growth of Gram-positive and Gram-negative bacteria, but had no antimicrobial activity to fungi. Bacteriocins produced by strain L1 and L17 only had antifungal activity (Hassan & Bullerman, 2008). To our knowledge, this is the first report of bacteriocin produced by L. paracasei subsp. tolerans that has significant antibacterial activity against both fungi and bacteria, especially Gram-negative bacteria. We had put the lyophilization bacteriocin F1 at room temperature and found its antimicrobial activity was stable. These results show that bacteriocin F1 may be a novel bacteriocin with a potential application in the food industry.