Abstract: Mycelium-sand composite lightweight soil (MBLS) is a lightweight geotechnical material composed of fungal mycelium, wheat bran, and sand. It has attracted significant attention due to its lightweight and environmentally friendly properties. While much research has focused on its macroscopic mechanical properties, studies on its microscopic characteristics remain limited. This paper investigates the strength formation mechanism of MBLS using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray energy spectroscopy. The results indicate that fungal colonization did not generate new mineral species, with only aragonite and calcite present. However, changes were observed in mineral content, crystallite size, and crystallinity, with distinct patterns of change for aragonite and calcite. During colonization, oxidation reactions occurred between the fungus and the particles, resulting in significant alterations to the functional groups on the sample surfaces. This process led to the formation of polysaccharides, ester compounds, and inorganic products, which enhanced the hydrophobicity of the particles. The mycelium adhered to and penetrated the pores and cracks on the surface of calcareous sand, dissolving existing calcium carbonate minerals and forming aragonite-type calcium carbonate. The strength of the MBLS specimens resulted from the combined effects of biochemical and biophysical processes. Polar groups from the fungal cell walls and extracellular polymers initially bonded with the negatively charged surfaces of the particles and metal cations through hydrogen bonding, adsorption, co-precipitation, and crystallization, forming microaggregates. The growth of the mycelium applied pressure and traction on the particles, enlarging the aggregates. The mycelium then linked the aggregates and filled the voids, ultimately forming the mycelium-sand composite lightweight soil. This study provides insights for the development and application of environmentally friendly lightweight geotechnical materials.