Two new diketopiperazines (1, 2), one new polyprenol (3), together with 19 known compounds (4-22) were obtained from the EtOAc extract of Bionectria sp. Y1085, an endophytic fungus isolated from the plant Huperzia serrata. Their structures were elucidated by extensive NMR and MS analysis. Bionectin D (1) is a rare diketopiperazine with a single methylthio substitution at the α-carbon of cyclized amino acid residue. The antibacterial activity of compounds was assayed against Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium ATCC 6539, and some metabolites (1, 2, 10, 11, and 14) exhibited evident antibacterial activity.