A new metabolite containing a γ-butyrolactone and 2-hydroxy-3 formylaminobenzoic acid moieties, opantimycin A (1) was isolated from a microbial metabolite fraction library generated from Streptomyces sp. RK88-1355 by search of an LC/MS-based spectral database named NPPlot: Natural Products Plot. The structure of 1 was determined based on extensive spectroscopic methods including NMR, MS and MS/MS experiments. 1 showed moderate cytotoxicity against HL-60 cell lines and antimalarial activity against Plasmodium falciparum 3D7. It was speculated that 1 might be biosynthesized by a hybrid enzyme including non-ribosomal peptide synthetase and polyketide synthase, which were similar to neoantimycin biosynthetic machinery.