ABSTRACT The Burkholderia cepacia complex (Bcc) consists of 20 closely related Gram-negative bacterial species that are significant pathogens for persons with cystic fibrosis (CF). Some Bcc strains are highly transmissible and resistant to multiple antibiotics, making infection difficult to treat. A tailocin (phage tail-like bacteriocin), designated BceTMilo, with a broad host range against members of the Bcc, was identified in B. cenocepacia strain BC0425. Sixty-eight percent of Bcc representing 10 species and 90% of non-Bcc Burkholderia strains tested were sensitive to BceTMilo. BceTMilo also showed killing activity against Pseudomonas aeruginosa PAO1 and derivatives. Liquid chromatography-mass spectrometry analysis of the major BceTMilo proteins was used to identify a 23-kb tailocin locus in a draft BC0425 genome. The BceTMilo locus was syntenic and highly similar to a 24.6-kb region on chromosome 1 of B. cenocepacia J2315 (BCAL0081 to BCAL0107). A close relationship and synteny were observed between BceTMilo and Burkholderia phage KL3 and, by extension, with paradigm temperate myophage P2. Deletion mutants in the gene cluster encoding enzymes for biosynthesis of lipopolysaccharide (LPS) in the indicator strain B. cenocepacia K56-2 conferred resistance to BceTMilo. Analysis of the defined mutants in LPS biosynthetic genes indicated that an α- d -glucose residue in the core oligosaccharide is the receptor for BceTMilo. IMPORTANCE BceTMilo, presented in this study, is a broad-host-range tailocin active against Burkholderia spp. As such, BceTMilo and related or modified tailocins have potential as bactericidal therapeutic agents against plant- and human-pathogenic Burkholderia .