Metal contamination represents a critical environmental challenge, adversely impacting ecosystems and human health. Microorganisms, including fungi, have developed diverse mechanisms to tolerate and resist metal-induced stress, making them valuables for bioremediation. This study evaluates the metal tolerance of Absidia glauca, Penicillium bilaiae, and Trichoderma viridescens using minimum inhibitory concentration (MIC) assay and the alternative minimum profile change concentration (MPCC) approach via MALDI-TOF MS. MIC assay revealed species-specific tolerances to copper, zinc, and cadmium. A. glauca showed the highest tolerance to copper and cadmium (75 and 9 mg L-1), producing a dry biomass of 0.03 and 0.04 g, respectively. While P. bilaiae exhibited the highest tolerance to zinc (75 mg L-1) producing a dry biomass of 0.06 g. MALDI-TOF MS provided rapid proteomic information on fungal responses to metals, showing changes in the protein profile as the metal concentration increased. We performed a comparative analysis between the values obtained in the MIC and MPCC, giving a positive correlation in the results of both techniques for Cu, Zn, and Cd (r = 1.00; 0.87 and 0.99 respectively, p < 0.05). In conclusion, MALDI-TOF MS has proven to be an effective method for analyzing fungal proteomic responses to metal exposure, providing more detailed molecular insights than traditional MIC assays. Future studies should investigate the mechanisms underlying metal resistance, particularly focus on the regulation of specific proteins.