Diarrhea is a debilitating condition in HIV infected individuals and with the finding that almost 1/4 cases of diarrhea in HIV are due to microsporidia, there is a dire need to institute measures for its detection on a regular basis. Keeping this in mind the study aims to determine the burden of intestinal microsporidiosis in HIV seropositive patients presenting with and without diarrhea and to compare the ability of microscopy and PCR in its detection.The study group consisted of 120 patients divided into four groups HIV seropositive with/without diarrhea, and HIV seronegative with/without diarrhea. Performance of four staining techniques including Modified Trichrome, Calcofluor White, Gram Chromotrope and Quick hot Gram Chromotrope stains were evaluated against PCR in diagnosing enteric microsporidiosis from stool samples.Overall prevalence of intestinal microsporidiosis was 10.83%. The same for HIV seropositive patients with diarrhea was 23.33%, HIV seropositive patients without diarrhea and in immune-competent hosts with diarrhea was 10% each. Enterocytozoon bieneusi was found to predominate. Calcofluor white stain detected maximum microsporidia in stool samples (76.92%), followed by Modified Trichrome stain (61.5%), PCR (46.15%) and Gram Chromotrope and Quick hot Gram Chromotrope stains (38.4% each). PCR exhibited the best performance with a sensitivity and specificity of 100%. Our data suggests screening of stool samples with either Modified Trichrome or Calcofluor white stain followed by PCR confirmation thus leading to maximum detection along with speciation for complete cure.