Virulence of Clostridium difficile is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of C. difficile possessing only CDT genes (A−B−CDT+) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of C. difficile has been suggested previously. Most human diagnostic tests will not detect A−B−CDT+ strains of C. difficile because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A−B−CDT+ C. difficile in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (n = 43) were cultured by enrichment, and C. difficile PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 C. difficile isolates were recovered, including two A−B−CDT+ isolates. This corresponded to an A−B−CDT+ prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A−B− isolates, 2/3 CDT+ isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated C. difficile was found. Neither A−B−CDT+ isolate was the predominant A−B−CDT+ strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A−B−CDT+ C. difficile in patients and strain collections but the prevalence of human A−B−CDT+ C. difficile is rarely investigated. This study highlights the occurrence of A−B−CDT+ strains of C. difficile in symptomatic patients, warranting further investigations of its role in human infection.