Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B12 conjugates; namely the vitamin B12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B12 and PNA were tested, including a disulfide bond. We found that vitamin B12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)3K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B12 as a carrier of PNA oligonucleotides into bacterial cells.