This paper describes inhibition of Semliki Forest virus (SFV) replication by synthetic derivatives of naturally occurring triterpenoid betulin (1). Chemical modifications were made to OH groups at C-3 and C-28 and to the C-20−C-29 double bond. A set of heterocyclic betulin derivatives was also assayed. A free or acetylated OH group at C-3 was identified as an important structural contributor for anti-SFV activity, 3,28-di-O-acetylbetulin (4) being the most potent derivative (IC50 value 9.1 μM). Betulinic acid (13), 28-O-tetrahydropyranylbetulin (17), and a triazolidine derivative (41) were also shown to inhibit Sindbis virus, with IC50 values of 0.5, 1.9, and 6.1 μM, respectively. The latter three compounds also had significant synergistic effects against SFV when combined with 3′-amino-3′-deoxyadenosine. In contrast to previous work on other viruses, the antiviral activity of 13 was mapped to take place in virus replication phase. The efficacy was also shown to be independent of external guanosine supplementation.