One new phthalide (1) was isolated from aerial parts of Peperomia nivalis, along with known compounds (2 and 3), reported in this species for the first time. The structure of the new compound was characterised on the basis of 1D (1H and13C NMR), 2D (COSY, HMQC, HMBC and NOESY) NMR and high-resolution mass spectral (HRMS) data. Compound 2 was isolated from a natural source for the first time but previously synthesised. Compounds 1–3 were evaluated for their anti-Helicobacter pylori and anti-Plasmodium falciparum activities. Compound 1 showed moderate activities against H. pylori (MIC 47.5 μM) and the F32-Tanzania strain of P. falciparum (IC508.5 μM). Compounds 2 and 3 exhibited weak anti-H. pylori activity (MIC 241.3 and 237.6 μM, respectively) and were inactive against P. falciparum.