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AbstractMycelium is crucial in decomposing biomass and cycling nutrients in nature. While various environmental factors can influence mycelium growth, the role of substrate mechanics is not yet clear. In this study, we investigate the effect of substrate stiffness on mycelium growth. We prepared agar substrates of different concentrations to grow the mycelium, but kept other environmental and chemical conditions consistent. We made a time-lapse recording of the growing history with minimum interruption. We repeated our tests for different species. Our results generally support that mycelium grows faster on a stiffer substrate,Ganoderma lucidumgives the highest growth rate andPleurotus eryngiiis most sensitive to substrate stiffness. We combined experimental characterization and computational simulation to investigate the mechanism and discovered that mycelium concentrates on the surface of a rigid substrate, but penetrates the soft one. Our Monte Carlo simulations illustrate that such a penetration allows mycelium to grow in the three-dimensional space, but effectively slows down the surface occupation speed. Our study provides insights into fungal growth and reveals that the mycelium growth rate can be tuned through substrate stiffness, thus reducing the time for producing mycelium-based composites. Impact statementWe used agar substrates and tuned its stiffness to culture mycelium and compared tune its stiffness to culture mycelium and compare its growth in a well-controlled condition. Our results revealed that mycelium grows faster on stiffer substrates, thus fully occupying the petri dish surface more quickly. We repeated our study several times by testing four species,P. eryngii,G. lucidum,Trametes versicolor,and Flammulina velutipes,and the stiffest substrate always gives the highest mean growing rate than others. TheG. lucidumshows the highest spreading rate that is obtained on the stiffest substrate as 39.1 ± 2.0 mm²/h. We found that the mycelium on a soft substrate will grow into the substrate instead of spreading on the stiffer surface. Our Monte Carlo simulations further show that once the fibers grow into a three-dimensional substrate, its growth is slower than growing on a two-dimensional surface, providing a microscopic mechanism of the substrate stiffness effect. This study’s analysis of how substrate stiffness impacts mycelium growth is new, bridging a critical knowledge gap in understanding the relationship between substrate mechanics and fungal ecology. The knowledge from this study has a potential in accelerating sustainable manufacturing of mycelium-based composite by adjusting substrate mechanics. Graphical Abstract