Nitric oxide (NO) is considered an important signaling molecule implied in various different physiological processes, including nervous transmission, vascular regulation, and immune defence, as well as the pathogenesis of several diseases. NO reportedly also has an antiviral effect on several DNA and RNA virus families. The NO-mediated S-nitrosylation of viral and host (macro)molecules appears to be an intriguing general mechanism for the control of the virus life cycle. In this respect, NO is able to nitrosylate cysteine-containing enzymes (e.g., proteases, reverse transcriptase, and ribonucleotide reductase). Moreover, zinc-fingers and related domains present in enzymes (e.g., HIV-1-encoded integrase or herpes simplex virus type-1 heterotrimeric helicase-primase complex) or nucleocapsid proteins may be considered as NO targets. Also, NO may regulate both host (e.g., nuclear factor-kappaB) and viral-encoded (e.g., HIV-1 tat protein or Epstein-Barr virus Zta) transcriptional factors that are involved in virus replication. Finally, NO-mediated S-nitrosylation of cysteine-containing glycoproteins and hemagglutinin may also occur. Here, NO targets are summarised, and the molecular bases for the antiviral effect of NO are discussed.