The identification of microorganisms, specifically fungal species, is a fundamental process to understand the taxonomy, diversity, ecology, pathogenicity, and applications. The traditional identification techniques hold limitations of accuracy based on morphological and phenotypical characters only. However, biochemical approaches, such as enzymes and metabolite profiling, have profound contributions before molecular and phylogenetic analysis. The enzyme studies relate the species diversity with intracellular and extracellular enzymes involved in various functions such as pathogenicity, nutrients, metabolism, habitat, etc. Similarly, metabolites, including both primary and secondary, provide insights into closely related taxa with emphasis on ecological and genetic adaptations. Such detailed characters require advanced techniques, including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR) spectroscopy for metabolites. This review aims to provide the principles, techniques, applications, and advanced research areas related to enzymes and metabolites profiling. The integration of enzymatic and metabolomic markers is expected to play an increasingly important role in fungal systematics, diagnostics, and biodiversity assessment.