Among several anti-gene strategies, the use of DNA analogues such as Peptide Nucleic Acids (PNAs) represents a promising tool for the modulation of gene transcription thanks to their chemical and enzymatic stability and the absence of charges in their backbone. PNAs can downmodulate oncogene expression successfully. We have recently demonstrated that the 7-mer PNA was able to selectively bind the longest loop of the G-quadruplex formed upstream of the P1 promoter of the Bcl-2 gene. Results demonstrated the ability of PNA-coated OAd5 oncolytic vectors to load and transfect their PNA cargo with a high efficiency and, also the synergistic cytotoxic effect against human A549 and MDA-MB-436 cancer cell lines. We have also demonstrated that the synthesized PNA does not interact with the corresponding duplex. To improve the target specificity towards the Bcl-2 P1 promoter, we extended the length of the pyrimidine-rich PNA from seven to ten bases. Herein, the 10-mer PNA and its FITC labelled analogue were synthesized, and the interaction with the N10–19 tract of bcl2midG4 in double strand was investigated by PAGE, CD and CD melting experiments. The druggability of the new PNAs was supported by fluorescence microscopy, which showed that the FITC-PNA specifically enters the cell nuclei. Finally, cytotoxicity assays confirmed the biological activity of the new anti Bcl-2 PNA. Overall, the studies here reported provide the basis for the development of new PNA-based anticancer agents for the treatment of human cancers.