Seven new polyketides including a phenol (<b>1</b>), two diphenyl ethers (<b>2</b> and <b>3</b>), two depsidones (<b>4</b> and <b>5</b>), and two phthalides (<b>6</b> and <b>7</b>) were isolated from the fungus <i>Aspergillus unguis</i> PSU-MF16 along with 27 known compounds. Their structures were determined by extensive spectroscopic analysis. The absolute configurations of <b>1</b> and <b>4</b>-<b>7</b> were established using comparative analyses of calculated and experimental ECD spectra. Among the new metabolites, <b>2</b> exhibited the best antimicrobial activity against <i>Staphylococcus aureus</i>, methicillin-resistant <i>S. aureus</i>, and <i>Microsporum gypseum</i> with equal MIC values of 16 μg/mL. In addition, known emeguisin A displayed potent antimicrobial activity against <i>S. aureus</i>, methicillin-resistant <i>S. aureus</i>, and <i>Cryptococcus neoformans</i> with equal MIC values of 0.5 μg/mL, compared with the standard drugs, vancomycin and amphotericin B. The structure-activity relationship study of the isolated compounds for antimicrobial activity is discussed.