A new family of putative signaling molecules having a 2(5H)-furanone configuration has been described in this work. They were released during late exponential or stationary phase in different growth media by some gram-positive bacteria, such as Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus sanfranciscensis, Enterococcus faecalis, and a gram-negative species, i.e. Salmonella enterica. A pair of 2(5H)-furanones called furanones A and B occurred in all the conditioned media (CMs) of the species considered. These two molecules showed similar retention times and their spectral data shared the key fragments to include them in the 2(5H)-furanones family. However, some differences were observed in the mass fragmentation profiles. In particular the use of PCA analysis of all the mass fragments enabled the grouping of furanone A profiles of S. enterica, L. helveticus, L. plantarum, L. paraplantarum, L. sanfranciscensis and E. faecalis in one unique cluster with only few exceptions. On the other hand, the mass fragmentation profiles of furanone B of the major part of the species and strains could be grouped together and were differentiated from those of L. helveticus. The specific activity of cell-free supernatants of high density cultures of S. enterica confirmed that the release of active molecules, and specifically of furanones A and B, was cell density dependent. Moreover, a preliminary experiment suspending S. enterica cells into cell-free supernatants of L. helveticus previously exposed to an oxidative stress demonstrated that furanones A and B have a strong interspecific activity. In fact cell autolysis and cell envelope damages were observed with Scanning Electron Microscopy (SEM) in S. enterica.