Due to the steady rise of multidrug-resistant pathogenic bacteria worldwide, it is critical to develop novel antibacterial drugs. This article presents chimeric antisense oligonucleotides that inhibit the bacterial growth of Staphylococcus aureus, one of the most frequent causes of hospital-acquired infections. The chimeric antisense oligonucleotides have a combination of first- and second-generation chemical modification. To deliver the antisense oligonucleotides into a cell, we apply a cell-penetrating oligopeptide attached to them. We have performed complete bioinformatics analyses of the glmS ribozyme present in S. aureus and its essential role in the biochemical pathway of glucosamine-6-phosphate synthesis. Besides, we have analyzed the bacteria for alternative metabolic pathways, such as the nagA gene. The first antisense oligonucleotide explicitly targets the glmS riboswitch, while the second explicitly targets the nagA mRNA. We have evaluated that combined, the antisense oligonucleotides block the synthesis of glucosamine-6-phosphate entirely and inhibit the bacterial growth of S. aureus. However, the glmS riboswitch targeting the antisense oligonucleotide is sufficient to inhibit the growth of S. aureus with a MIC80 of 5 μg/mL. The glmS ribozyme is a very suitable target for antibacterial drug development with antisense oligonucleotides.