Vaccines are important in preventing disease outbreaks and controlling the spread of disease in a population. A variety of vaccines exist, including subunit, recombinant, and conjugate vaccines. Glycoconjugate vaccines have been an important tool to fight against diseases caused by a number of bacteria. Glycoconjugate vaccines are often heterogeneous. Vaccines of the future are becoming more rationally designed to have a defined oligosaccharide chain length and position of conjugation. Homogenous vaccines could play an important role in assessing the relationship between vaccine structure and immune response. This review focuses on recent advances in the chemoenzymatic production of defined bacterial oligosaccharides for vaccine development with a focus on Neisseria meningitidis and selected WHO-prioritized antibacterial resistant-pathogens. We also provide some perspective on future advances in the chemoenzymatic synthesis of well-defined oligosaccharides.