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Resistance to aminoglycosides (Ag) is mostly due to a wide variety of modifying enzymes. Spread of aac(6)-Ib among pathogenic bacteria is a growing concern as it generates resistance to the clinically important Ag amikacin (Ak). A possible strategy to overcome this problem is to silence aac(6)-Ib. Several 17 nucleotide RNA molecules complementary to the five single stranded regions of aac(6)-Ib mRNA, carrying the consensus sequence for RNAseP ACCA in its 3-end were designed. These External Guided Sequences (EGS) were assessed in vitro for their capacity to bind to aac(6)-Ib mRNA and their ability to direct RNAseP digestion of the messenger. These results led to the selection of five candidates to perform in vivo experiments. In vivo expression of EGS demonstrated that EGSA2 and EGSC3 were able to reduce Ak resistance, being the latter one the most effective as seen in growth curves. In this strain the mRNA level showed a 50% reduction. Degradation of EGS is a problem to further develop this technology. To face this we designed antisense compounds with the EGSC3 sequence using several non-hydrolysable nucleic acid analogs: phosphorothioates, 2-O-Methyl and Locked Nucleic Acids (LNA) derivatives. Their binding to the mRNA and their capacity direct RNAseP-mediated cleavage of mRNA was studied. Our results suggest that LNA derivatives are able to induce RNAse P cleavage.