Clostridium difficile is an important cause of diarrhea in horses, causing sporadic and epidemic disease of varying severity. This study evaluated the molecular characteristics of 48 C. difficile isolates recovered from diarrheic horses admitted to a veterinary hospital by using PCR-ribotyping and toxin gene profile. Additionally, feces were tested for the presence of C. difficile toxin A/B via enzyme immunosorbant assay (EIA) in 38 horses. The toxin genes tcdA, tcdB and cdtB were present in 27 (56.25%), 35 (72.91%) and 2 (4.1%) strains, respectively. Eight isolates (16.6%) were A(-)B(+) variants. Thirteen of forty-eight isolates (27.0%) did not posses any toxin genes (A(-)B(-)CDT(-)). A positive EIA result was reported in 17 (44%) of the cases. There was no association between the presence of different ribotypes or strains and toxin gene(s) profiles and the clinical outcome.