INTRODUCTION: Clostridioides difficile infection (CDI) is one of the hospital-acquired infections and the most common cause for antibiotic-associated diarrhoea. Documentation of CDI is difficult, and interpretation of diagnostic results often requires consultation with clinical microbiologists. The purpose of this study was to compare the results of the combination of glutamate dehydrogenase enzyme (GDH) and toxin assay with the polymerase chain reaction (PCR) results, in order to find if the combination could substitute for the expensive molecular tests. MATERIALS AND METHODS: The sample size was statistically calculated to be 30, using an SPSS software. Both GDH and toxin assay were simultaneously tested in all the randomly selected stool samples, by simple random sampling, and irrespective of the results, they were also tested for tcdB gene by PCR in the present study. All the samples were also plated onto Brazier's C. difficile agar and incubated anaerobically. RESULTS: The sensitivity and specificity of GDH (using PCR as gold standard) were found to be 100% and 76.47%, respectively, and the sensitivity and specificity of toxin enzyme immunoassay (EIA) assay (using PCR as gold standard) were found to be 66.67% and 92.86%, respectively. However, when the toxin-equivocal results were also considered as positive, the sensitivity of toxin EIA was found to be 100%. The overall agreeability, using Cohen's Kappa statistic between GDH and toxin detection by enzyme-linked fluorescence assay, showed that they had moderate and substantial agreement, respectively, when compared to PCR. CONCLUSIONS: In this study, each of the toxin negatives and positives was also PCR negative and positive, respectively. All the toxin-equivocal samples tested positive on PCR, so it is our conclusion that in the settings where they cannot be taken for further molecular testing, those samples be considered as harbouring toxigenic C. difficile.