Biofilm-forming bacteria have increasingly exhibited resistance to conventional antibiotics, posing a formidable challenge in their treatment. In this study, antisense RNA (asRNA), a molecular strategy targeting messenger RNA (mRNA) molecules, was exploited to downregulate specific genes involved in biofilm formation in Klebsiella pneumoniae strains originating from diverse sources and characterized by strong biofilm production. Sequencespecific antisense oligonucleotides consisting of 24 base pairs were precisely designed to target two crucial genes, PgaC and cAMP, involved in biofilm formation and regulation. Subsequently, these designed oligonucleotides were synthesized artificially and then subjected to amplification through polymerase chain reaction (PCR). Following this, the amplified products were cloned into an antisense RNA-expressing plasmid and subsequently introduced into Klebsiella pneumoniae strains. The evaluation of biofilm formation efficacy and the quantification of target gene expression were carried out after the induction of asRNA expression using arabinose. The findings demonstrated a substantial reduction in biofilm formation at both the genotypic and phenotypic levels within Klebsiella pneumoniae strains harboring the asRNA constructs. This study represents a pioneering endeavor, marking the first application of antisense technology to counter biofilm formation in Klebsiella pneumoniae. Furthermore, the outcomes of this research present a potential therapeutic approach for addressing biofilm-associated infections across a spectrum of bacterial pathogens.