This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma Acholeplasma laidlawii cultivated in five different cultiva-tion media. The influence of relevant filtration process parameters, in particular transmembrane pres-sure and filtration temperature, on their respective retention was tested. The impact of the filtration temperature was further evaluated for the Gram-negative bacteria species Brevundimonas diminuta, the Gram-positive bacteria species Staphylococcus epidermidis, the Pseudomonas phage PP7, and the mycoplasma species Mycoplasma orale. The findings were correlated to the different mechanical properties of the particles, especially also with respect to the different bacterial cell envelopes found in those species. This study suggests that mycoplasma, surrounded by a flexible lipid bilayer, are signifi-cantly susceptible to changes in temperature, altering the stiffness of the cell envelope. Mycoplasma retention could thus be increased significantly by a decreased filtration temperature. In contrast, Gram-negative and Gram-positive bacteria species, with a cell wall containing a cross-linked peptidoglycan layer, as well as bacteriophages PP7 exhibiting a rigid protein capsid, did not show a temperature-dependent retention within the applied filtration temperatures between 2 and 35 °C. The trends of the retention of A. laidlawii with increasing temperature and transmembrane pressure were independent of cultivation media. Data obtained with mycoplasma M. orale suggest that the trend of mycoplasma retention at different filtration temperatures is also independent of the membrane pore size and thus retention level.