The most important aspect of diagnosis is distinguishing between granuloma inguinale and other STDs, particularly since many patients will be infected with more than one STD. Public health officials recommend that patients tested for granuloma inguinale be given a blood test for syphilis as well. In addition, the doctor will need to distinguish between granuloma inguinale and certain types of skin cancer, amebiasis, fungal infections, and other bacterial ulcers. The most significant distinguishing characteristic of granuloma inguinale is the skin ulcer, which is larger than in most other diseases, painless, irregular in shape, and likely to bleed when touched.

The diagnosis of granuloma inguinale is made by finding Donovan bodies in samples of the patient's skin tissue. Donovan bodies are oval rod-shaped organisms that appear inside infected tissue cells under a microscope. The doctor obtains a tissue sample either by cutting a piece of tissue from the edge of an skin ulcer with a scalpel or by taking a punch biopsy. To make a punch biopsy, the doctor will inject a local anesthetic into an ulcerated area and remove a piece of skin about 1/16 of an inch in size with a surgical skin punch. The tissue sample is then air-dried and stained with Wright's stain, a chemical that will cause the Donovan bodies to show up as dark purple safety pin-shaped objects inside lighter-staining capsules.