Previously unreported anthraquinone, acetylpenipurdin A (4), biphenyl ether, neospinosic acid (6), dibenzodioxepinone, and spinolactone (7) were isolated, together with (R)-6-hydroxymellein (1), penipurdin A (2), acetylquestinol (3), tenellic acid C (5), and vermixocin A (8) from the culture of a marine sponge-associated fungus Neosartorya spinosa KUFA1047. The structures of the previously unreported compounds were established based on an extensive analysis of 1D and 2D NMR spectra as well as HRMS data. The absolute configurations of the stereogenic centers of 5 and 7 were established unambiguously by comparing their calculated and experimental electronic circular dichroism (ECD) spectra. Compounds 2 and 5–8 were tested for their in vitro acetylcholinesterase and tyrosinase inhibitory activities as well as their antibacterial activity against Gram-positive and Gram-negative reference, and multidrug-resistant strains isolated from the environment. The tested compounds were also evaluated for their capacity to inhibit biofilm formation in the reference strains.