Enterotoxigenic Bacteroides fragilis (ETBF) is an important aetiological agent of acute diarrhoeal illness in humans. It is a fastidious anaerobe and takes 3-5 days when grown in culture, and test for enterotoxin detection requires additional time conventionally. The present study was aimed to standardize a rapid and sensitive diagnostic assay and to screen the presence of ETBF isolates directly in faecal samples using polymerase chain reaction (PCR) technique.A total of 85 faecal samples were processed for both aerobic and anaerobic bacterial isolation by culture technique and also subjected to PCR, for amplification of neuraminidase and enterotoxin genes of B. fragilis using specific primers.PCR was found to be positive for neuraminidase gene in 14/65 (21.5%) and for both neuraminidase and enterotoxin genes in 4/65 (6.1%) faecal samples within 48 h in study group. B. fragilis did not grow in any of the faecal samples when cultured anaerobically. However, the delay in transport and processing of faecal samples for anaerobic culture may be responsible for culture negativity.PCR assay can be used as a rapid and sensitive diagnostic tool for the identification of ETBF in culture as well as directly from diarrhoeal faecal samples instead of using conventional technique. The association of ETBF in diarrhoeal diseases is unclear and importance of ETBF as a causal agent will require more studies in Indian population.