Four new tetramic acid analogues neopestalotins A-D (1-4), one new furanone derivative neopestalotin E (6), and the known compound hymenosetin have been isolated from the solid cultures of the plant endophytic fungus Neopestalotiopsis sp. The structures of the new compounds were determined mainly by nuclear magnetic resonance (NMR) experiments. The absolute configurations of 1 and 2 were assigned by circular dichroism (CD) data, whereas those of 3 and 4 were deduced by a combination of CD and heteronuclear long range coupling (HETLOC) data. Compound 2 showed modest antimicrobial activity against the Gram-positive bacteria, Bacillus subtilis, Staphylococcus aureus col, and Streptococcus pneumoniae.