BACKGROUND: Peptoniphilus vaginalis, a Gram-positive anaerobic coccus, is a recently characterized species within the Peptoniphilus genus. No studies have detailed its features. This report documents the first case of P. vaginalis bloodstream infection in a patient with diabetic foot infection, with a review of existing literature. CASE PRESENTATION: A 55-year-old male presented with multiple skin ulcers on both lower extremities and hyperpyrexia. Blood cultures were conducted, and analysis via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed two distinct colony morphologies, namely Peptoniphilus spp. and Staphylococcus epidermidis. Discrepancies between MALDI-TOF MS results and biochemical identification prompted 16S rRNA sequencing, which confirmed P. vaginalis. The patient underwent surgical debridement and received intravenous antibiotics (levofloxacin, linezolid, and ornidazole), resulting in favorable recovery. CONCLUSIONS: This is the first report of P. vaginalis bloodstream infection in a patient with diabetic foot infection. It was observed that conventional biochemical identification methods and MALDI-TOF MS technology may not reliably detect P. vaginalis. In the absence of more advanced molecular methods, P. vaginalis could potentially be misidentified as Peptoniphilus harei, even in the previously published literature. Catalase testing may serve as a pragmatic discriminator in resource-limited settings, which required further validation. This underscores the necessity for employing molecular biology techniques to ensure precise diagnosis and optimize treatment strategies.