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Understanding which carbon or nitrogen sources the competing fungal species prefer is pivotal for specific biotechnological applications. However, this is not straightforward, as each strain sometimes behaves differently under the experimental conditions adopted. To analyse the trophic overlap of two species, it is necessary to refine diagnostic techniques and exclude variables that may interfere with the measurements. A protocol for establishing the suitability of chromogenic dyes in the analysis of filamentous fungi with phenotype microarrays is described here. The research goal was to determine the most suitable redox dye indicator and its optimal concentration that reacts quantitatively to the respiratory activity of both Verticillium spp. and Trichoderma spp. isolates in the presence of a nitrogen source. The commercial BiologTM Redox Dye Mixes D, E, and F and also TTC (2,3,5-Triphenyltetrazolium chloride), INT (2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride), and MTT solutions were evaluated in selected concentrations. Both their nutritive and toxic effects were quantified. Results showed that 1% “F” and 0.5% “D” BiologTM dyes were appropriate for the functional evaluation of isolates belonging to the Verticillium and Trichoderma genera. They can be used in BiologTM PM-nitrogen plate respiratory assays for a comprehensive functional characterisation of these organisms. [ABSTRACT FROM AUTHOR]