Pradimicins A, B, C, D, E, FA-1 and FA-2 were previously reported to exhibit broad activity against pathogenic fungi and yeasts, sharing a 5,6-dihydrobenzo[a]naphthacenequinone common skeleton and differing in amino acid moiety and 4'-amino group of D-thomosamine in the disaccharide moiety. Benanomicin A, also with this skeleton, has D-alanine as the amino acid moiety and D-xylosyl-D-fucose as the C-5 disaccharide moiety. In the continued search for new antifungal compounds, a new Actinomadura strain R103-3 producing pradimicin L was discovered. Based on taxonomic studies, Actinomadura verrucosospora subsp. neohibisca subsp. nov. is proposed for strain R103-3. Pradimicin L, a new pradimicin A congener with a D-glucosyl-D-thomosamine moiety at the C-5 position, was isolated from this strain. Its structure was deduced by MS, NMR spectrometry and degradation studies as N-[[(5S;6S)-5-O-[4,6-dideoxy-4-(methylamino)-3-O-(β-D-glucopyranosyl)-β-D-galactopyranosyl]-5,6,8,13-tetrahydro-1,5,6,9,14-pentahydroxy-11-methoxy-3-methyl-8,13-dioxobenzo[a]naphthacene-2-yl]carbonyl]-D-alanine. Pradimicin FL, with a D-serine moiety instead of D-alanine, was produced by directed biosynthesis in D-serine-supplemented medium. Pradimicins L and FL have broad in vitro antifungal activity: Pradimicin L is equiactive to pradimicin A, and pradimicin FL is more active than pradimicin L. This paper deals with the taxonomy of strain R103-3, structure elucidation of pradimicins L and FL, and their antifungal evaluation.