Abstract Here we propose a software for the estimation of the occupied area and volume of fungal cultures. This software was developed using a Matlab platform and allows analysis of high‐definition images from optical, electronic or atomic force microscopes. In a first step, a single hypha grown on potato dextrose agar was monitored using optical microscopy to estimate the change in occupied area and volume. Weight measurements were carried out to compare them with the estimated volume, revealing a slight difference of less than 1.5%. Similarly, samples from two different solid‐state fermentation cultures were analyzed using images from a scanning electron microscope (SEM) and an environmental SEM (ESEM). Occupied area and volume were calculated for both samples, and the results obtained were correlated with the dry weight of the cultures. The difference between the estimated volume ratio and the dry weight ratio of the two cultures showed a difference of 10%. Therefore, this software is a promising non‐invasive technique to determine fungal biomass in solid‐state cultures.