This review is primarily intended for synthetic bio-organic chemists and enzymologists who are interested in new strategies in the design of virus inhibitors. It is an attempt to assess the importance of the enzymatic properties of L-nucleosides and their analogues, particularly those that are active against viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpes simplex virus (HSV), etc. Only data obtained with purified enzymes have been considered and discussed. The examined enzymes include nucleoside- or nucleotide-phosphorylating enzymes, catabolic enzymes, viral target enzymes and cellular polymerases. The enantioselectivities of these enzymes were determined from existing data and are significant only when a sufficient number of enantiomeric pairs of substrates could be examined. The reported data emphasize the weak enantioselectivities of cellular or viral nucleoside kinases and some viral DNA polymerases. Thus, cellular deoxycytidine kinase has a considerably relaxed enantioselectivity with respect to a large number of nucleosides or their analogues, and it occupies a strategic position in the intracellular activation of the compounds. Similarly, HIV-1 reverse transcriptase often has a relatively weak enantioselectivity and can be inhibited by the 5-triphosphates of a large series of L-nucleosides and analogues. In contrast, degradation enzymes, such as adenosine or cytidine deaminases, generally demonstrate strict enantioselectivities favouring D-enantiomers and are used by chemists in asymmetric syntheses. The weak enantioselectivities of some enzymes involved in nucleoside metabolism are more or less pronounced, and one enantiomer or the other is favoured depending on the substrate. This suggests that the low enantioselectivity is fortuitous and does not result from evolutionary pressure, since these enzymes do not create or modify asymmetric centres in substrates. The combined enantioselectivities of the enzymes examined in this review strongly suggest that the field of L-nucleosides and their analogues should be systematically explored in the search for new virus inhibitors.