Five new compounds were isolated from Penicillium sp. Y-5-2 including an austin derivative 4, four isocoumarins 9, 11, 12, and 13, together with two known isocoumarins 8 and 10, and six known austin derivatives 1, 2, 3, 5, 6, and 7 and one phenol 14. Their structures and relative configurations were established by spectroscopic means. The absolute configurations of 4, 11, and 13 were defined mainly by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. The cyclization of the pentan-2-ol pendant at C-3 in compound 13 allowed the assignment of a new 2,3,4,4a,6,10b-hexahydro-1H-benzo[c]chromene isocoumarin skeleton. New compounds 9, 11, and 13 revealed inhibitory activities against E. coli at MIC values around 32 μg/mL. The known compound 14 showed potent antibiotic activity against Staphylococcus aureus and Bacillus subtilis with MIC values 8 and 2 μg/mL, respectively, with no cytotoxicity when tested in vitro. A rapid and efficient technique for selecting antibiotic fungal strain among eight marine-derived fungi was also described.