The study investigated the antiretroviral efficacy, toxicity, and cellular uptake of phosphothioate (PS) oligonucleotides and PS/LNA-modified oligonucleotides against HIV-1 in MT-4 cell cultures. Oligonucleotides were designed to bind conserved regions of the HIV-1 genome and modified with LNA nucleotides at the 3' or 5' ends. The best viral inhibition (IC50 90 ± 10 nM) was achieved with the LNA-free PS oligonucleotide targeting the HIV-1 integrase coding region. Among the LNA constructs, the optimal effect was for the oligonucleotide with 5'-LNA modification on the integrase region (IC50 1.12 ± 0.03 µM). LNA modifications did not increase antiviral activity over pure PS oligonucleotides due to poorer cellular internalization. PS oligonucleotides showed better uptake (approximately 12% FAM+ cells at 48 hours, 98% with cytoplasmic signal) and low toxicity (>92% viable cells at 48 hours).