An evaluation of the Vitek 2 ANC card (bioMérieux, Marcy l'Etoile, France) was performed with 301 anaerobic isolates. Each strain was identified by 16S rRNA gene sequencing, which is considered to be the reference method. The Vitek 2 ANC card correctly identified 239 (79.4%) of the 301 clinical isolates to the genus level, including 100 species that were not represented in the database. Correct species identification was obtained for 60.1% (181/301) of the clinical isolates. For the isolates not identified to the species level, a correct genus identification was obtained for 47.0% of them (47/100), and 16 were accurately designated not identified. Although the Vitek 2 ANC card allows the rapid and acceptable identification of the most common clinically important anaerobic bacteria within 6 h, improvement is required for the identification of members of the genera Fusobacterium, Prevotella, and Actinomyces and certain Gram-positive anaerobic cocci (GPAC).