An 11‐year‐old spayed female Basset Hound was presented to the Colorado State University Veterinary Teaching Hospital for evaluation of a 7‐week history of intermittent collapse, waxing and waning lethargy, and hyporexia. Abdominal ultrasound revealed a 6‐mm hypoechoic splenic nodule that, on cytologic evaluation, revealed marked neutrophilic inflammation with intracellular and extracellular bacterial rods frequently producing oval subterminal to terminal endospores, suggestive of Clostridium. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF) performed on bacteria isolated from this nodule initially identified a Clostridium species, which was eventually confirmed with 16 s rDNA sequencing. Computed tomography (CT) and exploratory laparotomy subsequently identified a 2.5‐cm diameter tubular structure beginning at the caudal aspect of the right kidney and coursing caudally containing gas and fluid, consistent with a retroperitoneal abscess, which was resected and also cultured Clostridium spp. The dog was discharged 3 days postoperatively and was alive at the time of writing, 7 months after discharge. This case highlights a previously unrecognized bacterial agent in a retroperitoneal abscess. The use of cytologic evaluation yielded a diagnosis of endospore‐forming bacteria suggestive of Clostridium sooner than culture and histopathology, which allowed for adjustment in the antibiotic protocol.