Since the discovery of a single-stranded RNA virus infectious to Rhizosolenia setigera (Bacillariophyceae), several novel diatom-infecting viruses have been isolated and examined as a potential mortality source for diatom populations. Here, we report the isolation and characterisation of a new single-stranded DNA (ssDNA) virus (CtenDNAV) that causes lysis of the cosmopolitan diatom species Chaetoceros tenuissimus (Meunier). The virion is 37 nm in diameter and accumulates in the nucleus of host cells. CtenDNAV has a closed circular ssDNA genome (5639 nt), which includes a partially double-stranded region (875 bp) and at least 3 major open reading frames (ORFs). One ORF is similar to putative replicase-related proteins of the previously reported ssDNA diatom viruses, CsalDNAV and CdebDNAV. On the basis of the genome structure and host range, CtenDNAV is considered to belong to the new genus Bacilladnavirus. CtenDNAV is the second C. tenuissimus virus; the first was the single-stranded RNA virus CtenRNAV. Data from the present study suggest that the C. tenuissimus natural population is affected by at least 2 viruses differing in genome type.