An actinomycete, strain B60(T), was isolated from a soil sample in Napier, Western Cape province of South Africa. Based on 16S rRNA gene sequence analysis and chemotaxonomy, strain B60(T) was identified as a member of the genus Actinomadura. Strain B60(T) produced an antibiotic with activity against Escherichia coli, Enterococcus faecium and Mycobacterium aurum, but not against Mycobacterium tuberculosis. Significant differences in morphological and physiological characteristics indicate that strain B60(T) represents a novel species of the genus Actinomadura. The name Actinomadura napierensis sp. nov. is proposed. The type strain is B60(T) (=DSM 44846(T)=NRRL B-24319(T)).