Biological phenotypes are important characteristics of microorganisms, and often reflect their genotype and genotype changes. Traditionally, Trichophyton rubrum (T. rubrum) phenotypes were detected using carbon source assimilation tests, during which the types of tested substances are limited. In addition, the operation is complicated, and only one substance can be tested at once. To observe the changes of the metabolic phenotype of T. rubrum after laser irradiation, a high-throughput phenotype microarray system was used to analyze the metabolism of different carbon, nitrogen, phosphorus and sulfur source substrates in a Biolog metabolic phenotyping system.The strain of T. rubrum used in this study can effectively utilize 33 carbon, 20 nitrogen, 16 phosphorus, and 13 sulfur source substrates prior to laser irradiation. After laser irradiation, the strain was able to utilize 10 carbon, 12 nitrogen, 12 phosphorus, and 8 sulfur source substrates. The degree of utilization was significantly decreased compared with the control. Both groups efficiently utilized saccharides and organic acids as carbon sources as well as some amino acids as nitrogen sources for growth. The number of substrates utilized by T. rubrum after laser irradiation were significantly reduced, especially carbon substrates. Some substrates utilization degree in the laser treated group was higher than control, such as D-glucosamine, L-glutamine, D-2-Phospho-Glyceric Acid, D-glucosamine-6-phosphate, and D-methionine.Laser irradiation of T. rubrum may lead to changes in the metabolic substrate and metabolic pathway, thus weakening the activity of the strain.© 2023. The Author(s).