Recent discovery of potent antibacterial antisense PNA-peptide conjugates encouraged development of a fast and efficient synthesis protocol that facilitates structure-activity studies. The use of an Fmoc/Boc protection scheme for both PNA monomers and amino acid building blocks in combination with microwave-assisted solid-phase synthesis proved to be a convenient procedure for continuous assembly of antisense PNA-peptide conjugates. A validated antisense PNA oligomer (CTCATACTCT; targeting mRNA of the acpP gene) was linked to N-terminally modified drosocin (i.e., RXR-PRPYSPRPTSHPRPIRV; X = aminohexanoic acid) or to a truncated Pip1 peptide (i.e., RXRRXR-IKILFQNRRMKWKK; X = aminohexanoic acid), and determination of the antibacterial effects of the resulting conjugates allowed assessment of the influence of different linkers as well as differences between the L- and D-forms of the peptides. The drosocin-derived compound without a linker moiety exhibited highest antibacterial activity against both wild-type Escherichia coli and Klebsiella pneumoniae (MICs in the range 2-4 μg/mL ∼ 0.3-0.7 μM), while analogues displaying an ethylene glycol (eg1) moiety or a polar maleimide linker also possessed activity toward wild-type K. pneumoniae (MICs of 4-8 μg/mL ∼ 0.6-1.3 μM). Against two colistin-resistant E. coli strains the linker-deficient compound proved most potent (with MICs in the range 2-4 μg/mL ∼ 0.3-0.7 μM). The truncated all-L Pip1 peptide had moderate inherent activity against E. coli, and this was unaltered or reduced upon conjugation to the antisense PNA oligomer. By contrast, this peptide was 8-fold less potent against K. pneumoniae, but in this case some PNA-peptide conjugates exhibited potent antisense activity (MICs of 2-8 μg/mL ∼ 0.3-1.2 μM). Most interestingly, the antibacterial activity of the D-form peptide itself was 2- to 16-fold higher than that of the L-form, even for the colistin- and tigecycline-resistant E. coli strains (MIC of 1-2 μg/mL ∼ 0.25-0.5 μM). Low activity was found for conjugates with a two-mismatch PNA sequence corroborating an antisense mode of action. Conjugates containing a D-form peptide were also significantly less active. In conclusion, we have designed and synthesized antisense PNA-drosocin conjugates with potent antibacterial activity against colistin- and tigecycline-resistant E. coli and K. pneumonia without concomitant hemolytic properties. In addition, a truncated D-form of Pip1 was identified as a peptide exhibiting potent activity against both wild-type and multidrug-resistant E. coli, P. aeruginosa, and A. baumannii (MICs within the range 1-4 μg/mL ∼ 0.25-1 μM) as well as toward wild-type Staphylococcus aureus (MIC of 2-4 μg/mL ∼ 0.5-1.0 μM).