Foot-and-mouth disease virus (FMDV) is an important pathogen that affects livestock breeding and causes huge economic losses worldwide. Currently, the development of antiviral agents to combat FMDV infection at the early stages is being explored. As viral replication critically depends on the host for nucleoside supply, host enzymes involved in nucleotides biosynthesis may represent potential targets for the development of antiviral agents. In the present study, the effects of IMP dehydrogenase (AVN-944 and mycophenolate mofetil) and dihydroorotate dehydrogenase (teriflunomide) inhibitors were evaluated both in vitro and in vivo. The results revealed that these compounds were effective in suppressing FMDV (O/MY98/BY/2010 and A/GD/MM/2013) infection. With regard to the antiviral mechanism, time-of-addition experiments revealed that these compounds were effective when added at the early stages of viral lifecycle (0–8 h post infection). However, exogenous guanosine/uridine eliminated the antiviral activity of these compounds. Importantly, treatment AVN-944 and teriflunomide significantly improved the survival of mice that were subcutaneously treated with FMDV. Together, the results of the present study indicate the broad-spectrum activities of anti-FMDV agents targeting IMP dehydrogenase or dihydroorotate dehydrogenase, which could be useful in developing strategies to prevent FMD.