Fourteen azaphilone-type polyketides (<b>1</b>-<b>14</b>), including nine new ones (<b>1</b>-<b>6</b> and <b>8</b>-<b>10</b>), were isolated from cultures of <i>Vitex rotundifolia</i>-associated <i>Penicillium</i> sp. JVF17, and their structures were determined by spectroscopic analysis together with computational methods and chemical reactions. Neuroprotective effects of the isolated compounds were evaluated against glutamate-induced neurotoxicity. Treatment with compounds <b>3</b>, <b>6</b>, <b>7</b>, and <b>11</b>-<b>14</b> increased cell viabilities of hippocampal neuronal cells damaged by glutamate, with compound <b>12</b> being the most potent. Compound <b>12</b> markedly decreased intracellular Ca<sup>2+</sup> and nuclear condensation levels. Mechanistically, molecular markers of apoptosis induced by treatment with glutamate, i.e., phosphorylation of MAPKs and elevated Bax/Bcl-2 expression ratio, were significantly lowered by compound <b>12</b>. The azaphilones with an isoquinoline core structure were more active than those with pyranoquinones, but <i>N</i>-substitution decreased the activity. This study, including the structure-activity relationship, indicates that the azaphilone scaffold is a promising lead toward the development of novel neuroprotective agents.