Burrowing mammals such as European sousliks are widespread and contribute significantly to soil ecosystem services. However, they have declined across their range and the non-invasive estimation of their actual population size has remained a challenge. Results support that the number of burrow entrances is positively correlated with population abundance, and burrow locations indicate the occupied area. We present an imagery-based method to identify and count animals’ burrows semi-automatically by combining remotely recorded red, green, and blue (RGB) images, pixel-based imagery, and random forest (RF) classification. Field images were collected for four colonies, then combined and processed by histogram matching and spectral band normalization to improve the spectral distinctions among the categories BURROW, SOIL, TREE, and GRASS. The accuracy indexes of classification for BURROW kappa (κ) were 95% (precision) and 90% (sensitivity). A 10-iteration bootstrapping of the final model resulted in coefficients of variation (CV%) of BURROW κ for sensitivity and precision lower than 5%; moreover, CV% values were not significantly different between those scores. The consistency of classification and balanced precision and sensitivity confirmed the applicability of this approach. Our approach provides an accurate, user-friendly, and relatively simple approach to count the number of burrow openings, estimate population abundance, and delineate the areas of occupancy non-invasively.