A new approach integrating micro X-ray fluorescence (μ-XRF) and digital image processing is proposed to quantify the spatial distribution of calcium carbonate crystals for microbial-induced calcite precipitation (MICP)-treated sand. Scanning the entire MICP-treated sample slice with the μ-XRF imaging method presents an overview of the distribution of Si and Ca elements, corresponding to the distributions of quartz sand and calcium carbonate particles, respectively. To improve the accuracy of the μ-XRF-imaging technique, scanning electron microscope tests were conducted and used as a basis for calibrating the segmentation threshold. μ-XRF images reveal that MICP effectively reduces the soil porosity and enhances the inter-particle contacts. Various quantification indexes (including area proportion, apparent porosity, apparent calcium carbonate content and pore filling rate) obtained through quantitative analysis of the μ-XRF image highlight the heterogeneous distribution of calcite precipitations at inter-particle contacts. The advantages and limitations of μ-XRF imaging technique were discussed, revealing its potential and further improvements needed for efficient, large-scale bio-cemented soil characterizations. This study presents a new approach to microstructural characterization of bio-cemented soils, and provides microscopic insights into the governing bio-cementation mechanisms.