As anthropogenic activities continue to introduce various contaminants into the environment, the need for effective monitoring and bioremediation strategies is critical. Fungi, with their diverse enzymatic arsenal, offer promising solutions for the biotransformation of many pollutants. While conventional research reports on ligninolytic, oxidoreductive, and cytochrome P450 (CYP) enzymes, the vast potential of fungi, with approximately 10 345 protein sequences per species, remains largely untapped. This review describes recent advancements in fungal proteomics instruments as well as software and highlights their detoxification mechanisms and biochemical pathways. Additionally, it highlights lesser-known fungal enzymes with potential applications in environmental biotechnology. By reviewing the benefits and challenges associated with proteomics tools, we hope to summarize and promote the studies of fungi and fungal proteins relevant in the environment.