Peptide nucleic acids (PNAs) are synthetic molecules that are like DNA/RNA, but with different building blocks. They have been studied as potential antibiotics because they can specifically target and bind to mRNAs and disrupt the function of a targeted gene. This systematic review aims to provide an in-depth analysis of the current state of PNAs as antibacterial agents, define the characteristics of the effective PNA constructs, and address the gap in the PNA advancement to be clinically competent agents. Following the PRISMA model, we searched four electronic databases: Web of Science, PubMed, SciFinder and Scopus, and found 627 published articles between 1994 and 2023. After screening and a rigorous selection process using explicit inclusion and exclusion criteria, 65 scientific articles were selected, containing 656 minimum inhibitory concentration (MIC) data. The antibacterial activity of the PNAs was assessed against 20 bacterial species. The most studied Gram negative and Gram positive were Escherichia coli (n=266) and Staphylococcus aureus (n=53), respectively. Additionally, we revealed the effect of PNA design such as the construct length, binding location, and carrier agents on their antibacterial activities. Finally, we examined the effects of antibacterial test models to assess PNA's inhibitory effects, emphasizing gaps and prospects. This systematic review provides a comprehensive assessment of the potential of PNAs as antibacterial agents and offers valuable insights for researchers and clinicians seeking novel therapeutic strategies to combat antibiotic-resistant bacteria.