To evaluate biomass distribution in heterogeneous biofilms from their microscope images, it is often necessary to perform image thresholding by converting the gray-scale images to binary images consisting of a foreground of biomass material and a background of interstitial space. The selection of the gray-scale intensity used for thresholding is arbitrary but under the control of the operator, which may produce unacceptable levels of variability among operators. The quality of numerical information extracted from the images is diminished by such variability, and it is desirable to find a method that improves the reproducibility of thresholding operations. Automatic methods of thresholding provide this reproducibility, but often at the expense of accuracy, as they consistently set thresholds that differ significantly from what human operators would choose. The performance of five automatic image thresholding algorithms was tested in this study:​ (1) local entropy;​ (2) joint entropy;​ (3) relative entropy;​ (4) Renyi's entropy;​ and (5) iterative selection. Only the iterative selection method was satisfactory in that it was consistently setting the threshold level near that set manually. The extraction of feature information from biofilm images benefits from automatic thresholding and can be extended to other fields, such as medical imaging.