Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is one of the major causes of nosocomial infections. In addition to its physiological adaptation capacity, it can develop resistance to disinfectants and antibiotics through various mechanisms. Recently, new eradication methods are gaining attention. Therefore, in this study, an LNA-2′-O-methyl hybrid antisense oligonucleotide targeting the acyl carrier protein P (acpP) gene was introduced into P. aeruginosa isolates. The design was determined through sequence analysis and prediction of the secondary structure of mRNA by software. Niosomes were used for enhancing cellular uptake. The control of the binding and transfection ability of the sequence was determined fluorometrically by labeling with 6-Fam. The effects were determined with broth microdilution method and qPCR studies. Eight different formulations were prepared. Among these, one formulation has shown to have ASO complexation ability whose composition was 312 μl Span 80 + 69.5 mg Cholesterol+ 36.4 mg CTAB+1 ml Chloroform and 5 ml dH₂O. Thus this formulation was determined as the delivery system for the next stages. Significant gene inhibition was detected at the six isolates. Results of this study suggested that niosomes can be used as a delivery system for cellular uptake of ASO and could eliminate bacterial growth.