As part of our ongoing collaborative effort to discover potential anti-HIV-1 agents from plants, the CH2Cl2 extract of Calocephalus brownii F. Muell. was phytochemically investigated, which resulted in the isolation of two sesquiterpene lactones of the cis-fused guaianolides type, one new identified as 1α-hydroxy-3βH-3α,4α-epoxy-4,10-dimethyl-5αH,7αH,8βH,10αH-guai-11(13)-ene-7,8-olide (1) and one known identified 1αH-4α-hydroxy-4,10-dimethyl-5αH,7αH,8βH,10αH-guai-11(13)-ene-7,8-olide (2). Their structures were elucidated on the bases of IR, UV, 1D-NMR, 2D-NMR, DIFNOE and high-resolution ESI-TOF-MS. The anti-HIV-1 activity was evaluated and revealed that STLs (1 and 2) caused a reduction for the viability of mock-infected MT-4 cells by CC50 = 29.1 and 0.5 μg/mL, respectively, and caused 50% protection of MT-4 cells against HIV-1 induced cytopathogeneticy by EC50 = 29.1 and 0.5 μg/mL, respectively, as compared with Efavirenz (EFV) as positive control that showed a CC50 = 11.6 and EC50 = 0.0006 μg/mL, using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.