Actinomyces neuii can grow under aerobic culture conditions and shows a gram-positive rod morphology, similar to that of Corynebacterium spp. A. neuii is usually detected in local pus samples, and published cases of A. neuii bloodstream infections are rare. Here, we report a case of bloodstream infection caused by A. neuii subsp. anitratus. A 53-year-old woman with fever and hypotension was referred to our hospital. The patient underwent surgery for breast cancer and received chemotherapy after central venous (CV) port placement. On day 2, a blood culture in an anaerobic bottle yielded positive results, and A. neuii subsp. anitratus was identified via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) and 16S rRNA sequencing. The patient was diagnosed with bloodstream infection caused by A. neuii subsp. anitratus with CV port infection. The CV port was removed and antibiotic treatment resulted in symptom improvement so the patient was discharged on day 28 of hospitalization. MALDI–TOF MS and 16S rRNA sequencing were found to be more useful for the identification of A. neuii than for phenotypic identification. Further research on A. neuii subsp. anitratus infections is required to avoid delayed or missed diagnoses.