The single enantiomers of seven hydroxyl-substituted biphenyl-diarylpyrimidines were designed and synthesized by a bioisosterism strategy as novel HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). The cellular and enzymatic assays indicated that the novel obtained compounds had significant activities and relatively low cytotoxicity. The supercritical fluid chromatography (SFC) enantioseparations of the racemic compounds and the enantiomeric profiling resulted that the (S) forms were generally more potent than the (R) counterparts. Among all the chiral derivatives, (S)-(-)-12a showed the best potency with the antiviral activities against wild-type (WT) and single mutant strains (L100I, K103 N, Y181C, E138K; especially Y188L), and RT enzyme in the low nanomolar concentration range. Toward double mutant virus strains (F227L + V106A, RES056), (S)-(-)-12a possessed submicromolar antiviral activities. In addition, (S)-(-)-12a showed a high cell-based selectivity index (SI = 5822) and no apparent toxicity was observed in the in vivo acute toxicity assay and electrocardiogram. The molecular docking studies predicted the binding modes of the compounds with RT and explained the activity differences for the enantiomers. Although the rat pharmacokinetic assay indicated a poor oral metabolism of the hydroxyl compound, the promising antiviral activity of the chiral hydroxyl-substituted biphenyl-diarylpyrimidines provided valuable lead compounds for further anti-HIV drug design.