Sulfhydryl oxidases (SOX) are FAD-dependent enzymes capable of oxidising free thiol groups and forming disulphide bonds. Although the quantity of scientific papers and suggested applications for SOX is constantly increasing, only a limited number of microbial SOX have been reported and are commercially available. Hence, the aim of this study was to develop a fast and reliable qualitative plate test for screening novel secreted fungal SOX. The screening was based on the Ellman's reagent, i.e. 5,5′-dithiobis[2-nitrobenzoic acid]. Altogether, 32 fungal strains from an in-house culture collection were screened. A total of 13 SOX-producing strains were found positive in the plate test screen. The novel SOX producers were Aspergillus tubingensis, Chaetomium globusum, Melanocarpus albomyces, Penicillium aurantiogriseum, Penicillium funiculosum, Coniophora puteana and Trametes hirsuta. Six of the discovered SOX were partially characterised by determination of isoelectric point, pH optimum and substrate specificity. A. tubingensis was identified as the most efficient novel SOX producer.