A fungal biofilm refers to the agglomeration of fungal cells surrounded by a polymeric extracellular matrix (ECM). The ECM is composed primarily of polysaccharides that facilitate strong surface adhesion, proliferation, and cellular protection from the surrounding environment. Biofilms represent the majority of known microbial communities, are ubiquitous, and are found on a multitude of natural and synthetic surfaces. The compositions, and in-turn nanomechanical properties, of fungal biofilms remain poorly understood, because these systems are complex, composed of anisotropic cellular and extracellular material, and importantly are species and environment dependent. Therefore, genomic variation, and/or mutations, as well as environmental and growth factors can change the composition of a fungal cell's biofilm. In this work, we probe the physico-mechanical and biochemical properties of two fungal species, Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans), as well as two antifungal resistant sub-species of C. neoformans, fluconazole-resistant C. neoformans (FlucRC. neoformans) and amphotericin B-resistant C. neoformans (AmBRC. neoformans). A new experimental methodology of characterization is proposed, employing a combination of atomic force microscopy (AFM), instrumented nanoindentation, and Synchrotron ATR-FTIR measurements. This allowed the nano-mechanical and chemical characterisation of each fungal biofilm.