Ferrari, C.; Dehkohneh, A.; Schumacher, J.; Ogawa, Y.; Gerrits, R.; Fratzl, P.; Gorbushina, A. A.; Bidan, C. M.

Black extremotolerant fungi form persistent biofilms on a wide range of natural and engineered substrates. Able to weather minerals, affect stone monument surfaces, and colonize solar panels, they demonstrate a strong capacity to interact with and modify material surfaces, even under most extreme conditions. In this study, we establish a methodological workflow for the structural and mechanical characterization of melanized biofilms formed by the black fungus Knufia petricola. This species represents a broader group of resilient surface colonizers and provides a model for in-depth investigation. When grown on solid agar/air interface, this species predominantly forms a compact biofilm, composed of spherical cells, while retaining the capacity for filamentous growth, providing a suitable framework to explore morphology-dependent biomechanical responses. The proposed toolbox combines complementary analytical techniques spanning multiple spatial scales, including shear-rheology to quantify bulk viscoelastic behavior, micro-indentation to resolve local stiffness of the biofilm surface, micro-computed tomography for non-destructive three-dimensional visualization of biofilm architecture, and cryogenic preparation methods and electron microscopy for high-resolution ultrastructural analysis. As a case study, we applied this workflow to compare biofilms grown on two nitrogen sources (NO3- vs. NH4+). Our results reveal that the nitrogen source plays a key role in biofilm morphology across multiple hierarchical levels - ranging from cell division patterns and distribution of extracellular polymeric substances (EPS) to overall mechanical properties, where NO3- leads to budding-dominated growth and increased stiffness, whereas NH4+ promotes meristematic growth and softer biofilms. The successful transfer and integration of methods originally developed for bacterial biofilm research highlights the feasibility of quantitative mechanical analyses in fungal systems. This multiscale toolbox provides a foundation for advancing the mechanistic understanding of fungal biofilms and biofilm-material interactions, with implications for geomicrobiology, material biodeterioration, and the design of bio-inspired functional materials.