The ethyl acetate extracts prepared from the mycelia of three endophytic fungi Purpureocillium lilacinum, Aspergillus sp., and Fusarium sp., isolated from the roots of Rauvolfia macrophylla (Apocynaceae) were screened for their antiprotozoal activity in vitro against Plasmodium falciparum (NF54), Leishmania donovani , Trypanosoma brucei rhodesiense, and Trypanosoma cruzi . Amongst these extracts, the one from P. lilacinum showed potent antileishmanial activity against L. donovani (IC 50 value of 0.174 μg mL −1 ) with good selectivity (SI=94.9) toward the L6 cell line, whereas the other extracts were inactive and not selective. The fractionation and purification of the active extract from P. lilacinum by column chromatography over silica gel yielded a new ergochromone derivative ( 1 ), together with six known compounds: (22 E ,24 R )-stigmasta-5,7,22-trien-3- β -ol ( 2 ), (22 E ,24 R )-stigmasta-4,6,8(14),22-tetraen-3-one ( 3 ), emodin ( 4 ), chrysophanol ( 5 ), aloe-emodin ( 6 ), and palmitic acid, whose structures were elucidated spectroscopically. Compound 1 was tested in vitro for its antiparasitic activities against the above listed parasites and for its antimicrobial activity against Staphylococcus aureus , Bacillus cereus , Listeria monocytogenes , Escherichia coli , Providencia stuartii , Klebsiella pneumoniae , and Pseudomonas aeruginosa . The compound displayed potent antileishmanial activity against L. donovani with an IC 50 value of 0.63 μg mL −1 (0.87 μ m ) with good selectivity (SI=49.5) toward the L6 cell line. It also exhibited good antibacterial activity against three of the tested microbial strains B. cereus , E. coli ATCC879, and P. stuartii ATCC29916 with minimum inhibitory concentrations below 62.6 μg mL −1 . Compound 1 is thus a promising active compound that could be investigated for antileishmanial and antimicrobial drug development.