Provided herein is the first evidence that mycobacteriophage, indeed phage of any kind, have surface proteins (capsid and/or tail tube proteins) that are O-glycosylated. The glycosyltranferases that glycosylate these proteins are encoded in the phage genomes. In addition, for the three mycobacteriophages invested in this study, the glycans appear to be attached exclusively at a C-terminal serine residue. Each phage expresses a unique and novel O-glycan. The LC-MS files deposited here are the preliminary evidence that these mycobacteriophage proteins are glycosylated. A follow-up investigation will examine the composition and structure of the O-glycans in more detail.