Enhanced chemiluminescence (ECL) describes the phenomenon of the light output increase in the reaction of oxidation of luminol catalyzed by horseradish peroxidase (HRP) in the presence of certain phenolic compounds. This work summarizes the effects of preincubation of certain substances with HRP on the chemiluminescent reaction intensity. Preincubation of herbicide, detergent, surfactants (Brij-96 and Tween-20), phenol, metal ions (mercury, cobalt, and nickel), and bactericide with HRP had an inhibitory effect on the enzyme activity. HRP-preincubation with metal ions (cadmium, magnesium, and zinc), as well as with some insecticides, stimulated the chemiluminescent intensity. Calibration graphs were obtained to demonstrate the possibility to determine the pollutant concentration. Light emission from the peroxidase catalyzed enhanced chemiluminescence is affected by a wide number of chemicals and, therefore, the method can be used for onsite monitoring of water quality. A rapid and simple assay to detect water contamination has been developed.