The construction industry urgently requires sustainable building materials. Mycelium-based composites (MBCs), a class of bio-based materials grown from fungal mycelium on organic substrates, have emerged as a promising insulation solution. A key challenge in MBC development is in understanding the inter-species variability and its role in thermal performance. Fungal species influence thermal properties and MBC viability, however, studies quantifying differences between a large range of species are lacking. Additionally, the accuracy of thermal conductivity measurements varies between methodologies, with some approaches being unsuitable for anisotropic bio-based materials including MBCs. Addressing these uncertainties is critical, as optimising fungal species requires accurate, and repeatable, measurement methods to facilitate validation and meaningful comparison of inter-laboratory data. This study addresses these gaps by evaluating the thermal conductivity of MBCs produced using 20 fungal strains across 19 species, employing both transient (Hot Disk) and steady-state (Heat Flow Meter) methods. The research investigates interspecies variability in thermal conductivity, the impact of density on thermal conductivity, and methodological differences. Thermal conductivity measurements revealed significant interspecies variability, demonstrating the influence of fungal selection on insulation properties. While the specimens consistently exhibited thermal conductivity values comparable to traditional insulation materials (average thermal conductivity measured using the Heat Flow Meter was (0.0405 ± 0.0004) W/mK), transient methods systematically overestimated values by an average of 51% relative to steady-state measurements, influenced by material anisotropy and density. These findings underscore the importance of measurement methodology in evaluating bio-based materials and provide practical insights into species selection for scalable, sustainable insulation materials.