Chemical investigation of a new endophytic fungus, Mycosphaerella sp. nov. strain F2140, associated with the foliage of the plant Psychotria horizontalis (Rubiaceae) in Panama, resulted in the isolation of cercosporin (1) and a new cercosporin analog (3) as the major components. The structures of minor compounds in the extract were elucidated by detailed spectroscopic analysis as 2-(2-butyl)-6-ethyl-3-hydroxy-6-methylcyclohex-2-ene-1, 5-dione (4), 3-(2-butyl)-6-ethyl-5-hydroxy-2-methoxy-6-methyl-cyclohex-2-enone (5), and an isomer of 5 (6). To study the influence of the hydroxy groups on the antiparasitic activity of cercosporin, compound 1 was acetylated to obtain derivative 2. The isolated compounds 1-6 were tested in vitro to determine their antiparasitic activity against the causal agents of malaria ( Plasmodium falciparum), leishmaniasis ( Leishmania donovani), and Chagas disease ( Trypanosoma cruzi). Cytotoxicity and potential anticancer activity of these compounds were evaluated using mammalian Vero cells and MCF7 cancer cell lines, respectively. Compounds 1 and 2 displayed high potency against L. donovani (IC₅₀ 0.46 and 0.64 μM), T. cruzi (IC₅₀ 1.08 and 0.78 μM), P. falciparum (IC₅₀ 1.03 and 2.99 μM), and MCF7 cancer cell lines (IC₅₀ 4.68 and 3.56 μM). Compounds 3-6 were not active in these assays at a concentration of 10 μg/mL.