In this study, eight natural isocoumarins (<b>1</b>-<b>8</b>) were isolated from a marine-derived <i>Exserohilum</i> sp. fungus. To explore their structure-activity relationship and discover potent antimalarial leads, a small library of 22 new derivatives (<b>1a</b>-<b>1n</b>, <b>2a</b>, <b>3a</b>-<b>3c</b>, <b>4a</b>-<b>4c</b>, and <b>7a</b>) were semisynthesized by varying the substituents of the aromatic ring and the aliphatic side chains. The natural compound (<b>1</b>) and three semisynthetic derivatives (<b>1d</b>, <b>1n</b>, and <b>2a</b>), possessing an all-<i>cis</i> stereochemistry, exhibited strong antiplasmodial activity with IC<sub>50</sub> values of 1.1, 0.8, 0.4, and 2.6 μM, respectively. Mechanism studies show that <b>1n</b> inhibits hemozoin polymerization and decreases the mitochondrial membrane potential but also inhibits <i>P. falciparum</i> DNA gyrase. <b>1n</b> not only combines different mechanisms of action but also exhibits a high therapeutic index (CC<sub>50</sub>/IC<sub>50</sub> = 675), high selectivity, and a notable drug-like profile.