Stool samples from 86 immunocompromised patients (51 human immunodeficiency virus (HIV)-infected patients and 35 patients with haematologic malignancies) were systematically screened for intestinal microspordiosis by microscopic examination and polymerase chain reaction (PCR) using universal primer V1/PMP2. Nine samples (10.5%) showed amplification with the predictive size of fragment (6 from HIV-infected patients and 3 from patients with myeloma). Only 5 out of them (all HIV-infected patients) were revealed positive by microscopy. By means of amplicons fragment size, species-specific primers (V1/EB450, V1/IS500) and sequencing, 3 microsporidia species were for the first time identified in Tunisia: Enterocytozoon bieneusi (3 isolates), Encephelitozoon intestinalis (2 isolates), and Encephalitozoon hellem (1 isolate). Systematic use of such sensitive and discriminative molecular tools will contribute to determining the true prevalence of microsporidiosis in Tunisia and to better management of infected immunocompromised subjects.