In the last two decades, antisense oligonucleotide technology has emerged as a promising approach to tackling various healthcare issues and diseases, such as antimicrobial resistance, cancer, and neurodegenerative diseases. Despite the numerous improvements in the structure and modifications of the antisense oligonucleotides (ASOs), there are still specific problems with their clinical efficacy and preclinical cytotoxicity results. To better understand the effects of the ASOs in this paper, we conducted many MTT assays to assess the general and specific cytotoxicity of four new chimeric ASOs in bacterial cells and human cell lines. We demonstrate the absence of inhibitory activity in the human pathogenic bacteria Staphylococcus aureus by non-specific ASOs. The pVEC-ASO1 and pVEC-ASO2 are designed to have no specific targets in S. aureus. They have only partial hybridization to the guanylate kinase mRNA. The pVEC-ASO3 targets UBA2 mRNA, a hallmark cancer pathology in MYC-driven cancer, while pVEC-ASO4 has no complementary sequences. We discovered some cytotoxicity of the non-specific ASOs in healthy and cancer human cell lines. The results are compared with two other ASOs, targeting specific mRNA in cancer cells. All ASOs are delivered into the cell via the cell-penetrating oligopeptide pVEC, which is attached to them. We draw a good correlation between the thermodynamic stability of ASO/target RNA and the toxicity effect in human cell lines. The data obtained signify the importance of thorough bioinformatic analysis and high specificity in designing and developing novel ASOs for safer therapeutic agents in clinical practice.