Torrubiellone E (1), a new N-hydroxypyridone alkaloid, was isolated from the spider pathogenic fungus Torrubiella longissima BCC 2022, together with the known compounds, torrubiellones A (2) and B (3), and JBIR-130 (4). Compound 1 exhibited antimalarial activity against Plasmodium falciparum K1 with an IC 50 value of 3.2 μg/mL, while it also showed weak cytotoxic activities.