Reverse transcriptase (RT) is essential for the replication of human immunodeficiency virus (HIV), and clinically approved RT inhibitors face challenges of significant side effects and drug resistance, creating a need for new agents with low toxicity. Herein, we describe the antiviral activities of both enantiomeric forms of novel optically active 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) and their 5-fluoro analogues (dOTFC), a class of 2,4-disubstituted 1,3-oxathiolane nucleosides that hybridize 4'-thio and isonucleoside structures. We evaluated their anti-HIV-1 activity in cord blood mononuclear cells (CBMCs) and multiple cell lines (MT-4, Jurkat, H9, U937, CEM), and anti-hepatitis B virus (HBV) activity in the transfected human hepatoma cell line 2.2.15. Results show that compounds 2, 3, and 4 are potent and selective inhibitors of HIV-1 replication in vitro, with no cytotoxicity at concentrations up to 500 μM. Compound 1 exhibits potent anti-HIV-1 activity but only modest anti-HBV activity (IC50 = 17.5 μM), which is 1000-fold less potent than lamivudine, and is more cytotoxic than AZT in Jurkat, U937, and CEM cells. Among the enantiomers, 1 is 3-4 fold more potent than 2-4 but less selective, while 2 displays superior selectivity. Compounds 3 and 4 have similar potency and no cytotoxicity, an unprecedented observation in nucleoside research. The 5-fluoro substituent has negligible effects on the potency and cytotoxicity of 2 and 4 but reduces the cytotoxicity of 3. In conclusion, we report the first examples of enantiomerically pure 4'-thio nucleoside analogues with potent and selective anti-HIV-1 activity in primary lymphocytes and cell lines. Further details on resistance profiles and cellular metabolism will be reported in forthcoming publications.