Image pre-processing is highly significant in automated analysis of microscopy images. In this work, non-uniform illumination correction has been attempted using the surface fitting method (SFM), multiple regression method (MRM), and bidirectional empirical mode decomposition (BEMD) in digital microscopy images of tuberculosis (TB). The sputum smear positive and negative images recorded under a standard image acquisition protocol were subjected to illumination correction techniques and evaluated by error and statistical measures. Results show that SFM performs more efficiently than MRM or BEMD. The SFM produced sharp images of TB bacilli with better contrast. To further validate the results, multifractal analysis was performed that showed distinct variation before and after implementation of illumination correction by SFM. Results demonstrate that after illumination correction, there is a 26% increase in the number of bacilli, which aids in classification of the TB images into positive and negative, as TB positivity depends on the count of bacilli. © Microscopy Society of America 2014.