Aims To determine the characteristics of the novel phage tail-like bacteriocin (PTLB) produced by Stenotrophomonas maltophilia S16. Methods and Results A screen to identify novel bacteriocins from a panel of 86 S. maltophilia strains was performed. We found that 62 tested S. maltophilia strains were sensitive to a PTLB, designated maltocin S16. In addition, 8 of 14 tested Escherichia coli strains were also susceptible to maltocin S16. Minimum inhibitory concentration determination confirmed that maltocin S16 had rather high bactericidal activity against both S. maltophilia and E. coli. Maltocin S16 was resistant to trypsin and proteinase K. Furthermore, lipopolysaccharide was found to be involved in the adsorption of maltocin S16. The gene cluster of maltocin S16 was consisted of 23 putative open reading frames. Phylogenetic analysis indicated that maltocin S16 represents a new class of PTLB in S. maltophilia. Conclusion Maltocin S16 was a novel PTLB with broad-spectrum and high bactericidal activity against S. maltophilia and E. coli. Significance and Impact of the Study Stenotrophomonas maltophilia is a multidrug-resistant opportunistic pathogen with increasing contributions to infectious disease morbidity and mortality. Maltocin S16 could be a promising alternative for antibiotics against S. maltophilia and E. coli infections.