Three new bioactive compounds, namely ( S )-tenellic acid B dimethyl acetal ( 1a ), (3 R ,3′ R/S )-isotalarone ( 2 ), and (3 R ,5 R )- cis -5-methyl-3-(2-oxobutyl)-dihydrofuran-2-one ( 3 ), were isolated from the terrestrial fungus Penicillium purpurogenum MM, together with 15 known metabolites: talaroflavone, pestalasin A, altenuene, penicillide, 3′- O -methyl-dehydroisopenicillide, rubralactone, tenellic acid B, diaporthin, butyrolactone 1, butyrolactone-V, 4-hydroxy-2-methoxyacetanilide, ergosterol, ergosterol peroxide, linoleic acid, and glycerol monolinoleate. The chemical structures of the three new compounds were confirmed by extensive one- and two-dimensional NMR and electron spray ionization high-resolution mass spectra measurements and by comparison with literature data. The absolute configurations of the new compounds, and of talaroflavone ( 4a ) and tenellic acid B ( 2b ), were determined by ab initio calculations of ECD, ORD, and NMR data. The antimicrobial and cytotoxic activities of the crude extract and of the isolated compounds were studied using a set of microorganisms and brine shrimp assay, respectively. The isolation and taxonomic characterization of P. purpurogenum MM is reported.