Staphylococcus aureus is an important zoonotic bacterial pathogen easily acquiring antibiotic resistance, which has been frequently associated with food poisoning and nosocomial infections in humans as well as mastitis in dairy cows. The spread and emergence of antibiotic resistant S. aureus require the development of new antibiotics and/or non-antibiotic based anti-infective strategies. Here we report a novel peptide nucleic acid (PNA) that can antisense the cytidine monophosphate (CMP) kinase of Staphylococcus aureus with antibacterial activity in vitro and in vivo. Based on the genome sequence of S. aureus N315, novel antisense PNA conjugates with a bacterial penetration peptide (KFF)3K-L- were synthesized to block the expression of the seven potentially essential genes (cmk, deoD, ligA, smpB, glmU, pyrH, and ftsA) and evaluated for their antibacterial properties. Our experimental analyses demonstrated that two peptide conjugated-PNAs (P-PNAs) targeting either cmk or deoD genes, Pjyh-cmk1 and Pjyh-deoD1, had the strongest growth inhibitory effects against S. aureus ATCC 29740 (a bovine mastitic milk isolate) in a dose-dependent manner. Using a translational fusion system with the lacZ reporter gene, such an inhibitory effect was due to the antisense effect against the target genes rather than the antigene effect. In vivo application of Pjyh-cmk1 resulted in the significant reduction of bacterial loads in the intraperitoneally infected mice with a sublethal dose of S. aureus at 20 hrs post-infection. Moreover, Pjyh-cmk1 could dramatically increase the survival rate of the mice lactated after intra-mammary infection. Taken together, our characterization of Pjyh-cmk1 demonstrated the bactericidal activity against S. aureus, an important zoonotic bacterial pathogen, as well as in vivo effectiveness. This is another demonstration implying possible application of the antisense P-PNA as an alternative anti-infective agent.