A series of 2,3-dideoxy (D2) and 2,3-didehydro-2,3-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N⁴-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N⁴-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N⁴-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N⁴-modified nucleoside for the D2-series was N⁴-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N⁴-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N⁴-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N⁴-modification especially in the D2-nucleoside series containing the N⁴-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5′-triphosphate of the parent compound (D-D4FC-TP) was formed from the N⁴-acyl-D4FC analogues in different cells, the levels of the 5′-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC₉₀), suggesting that a direct interaction of the triphosphates of these N⁴-acyl nucleosides was involved in the antiviral activity.