In this study, eight natural isocoumarins (1–8) were isolated from a marine-derived Exserohilum sp. fungus. To explore their structure–activity relationship and discover potent antimalarial leads, a small library of 22 new derivatives (1a–1n, 2a, 3a–3c, 4a–4c, and 7a) were semisynthesized by varying the substituents of the aromatic ring and the aliphatic side chains. The natural compound (1) and three semisynthetic derivatives (1d, 1n, and 2a), possessing an all-cis stereochemistry, exhibited strong antiplasmodial activity with IC₅₀ values of 1.1, 0.8, 0.4, and 2.6 μM, respectively. Mechanism studies show that 1n inhibits hemozoin polymerization and decreases the mitochondrial membrane potential but also inhibits P. falciparum DNA gyrase. 1n not only combines different mechanisms of action but also exhibits a high therapeutic index (CC₅₀/IC₅₀ = 675), high selectivity, and a notable drug-like profile.