Zymogram analysis is a useful tool for the identification of several enzymes. The present study was undertaken to investigate the efficiency gains from the characterization of pectic enzymes on zymograms by staining of pectin-agarose overlays using cetyl trimethyl ammonium bromide also known as cetrimide or CTAB. The method is based on the fact that the enzymatic hydrolysis of the pectic substrates included in the agarose matrix gel inhibited their precipitation by CTAB, leading to the appearance of cleared zones in front of the pectin hydrolases and lyases. Conversely, esterases led to the increase of pectin precipitation. Fungal pectinolytic enzymes were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to the zymogram detection technique, using two pectin substances, namely citrus pectin and polygalacturonic acid. Overall, the findings presented in the current study indicate that several elements (ions, salts, pH, temperature, chelators, and reducing agents) may significantly affect the results of zymogram analysis and can, therefore, be employed to enhance the discriminatory and operational potential of the analysis in terms of accurate discrimination between several pectinolytic activities involved and effective implementation of the purification procedures required in the process.