The current experimental study investigates the influence of latex microsphere particles' size on the assessment of their hydrophilic/hydrophobic character, using the method known as “Microbial Adhesion to Hydrocarbons” (MATH). Since bacteria surfaces often change according to the environment in which they find themselves, most of the experiments here were carried out using the calibrated latex microspheres Polybeads® and Yellow-green Fluoresbrite® (Polyscience) microspheres with diameters between 0.2 μm and 4.5 μm. All the beads had a density of ˜1.05 g/cm3. The first set of experiments was performed to adapt the procedure for measurements of water contact angles to microsphere lawns. It was found that all the microspheres tested were hydrophobic, when using a water contact angle of around 110-118°. However, wide differences were observed using the MATH method. The smaller microspheres (0.2 μm, 0.5 μm +/- 0.75 μm) exhibited a poor affinity to hexadecane, even after long contact times, suggesting a hydrophilic character. In contrast, larger microspheres quickly adhered to hexadecane, which is consistent with the values obtained for the water contact angles observed. These results suggest that, at least where hydrophobic particles are concerned, the MATH method is not suitable for the assessment of the hydrophobic character of particles with diameters of less than 1.0 μm. We lastly investigated whether the data obtained for Bacillus spores could also be affected by spore size. The hydrophobicity of spores of eight Bacillus strains was analysed by both MATH and contact angle. Some discrepancies were observed between both methods but could not be related their size (length or width).