Aims: To investigate the effects of formulation ingredients used in ophthalmic preparations on thiomersal activity against Staphylococcus aureus and Pseudomonas aeruginosa . Methods and Results: Minimum inhibition concentrations (MICs) of the tested ingredients and their combinations were studied using partial broth dilution checkerboard method. Complex formation was determined using differential scanning calorimetry (DSC) and u.v. scan. Isotonic agents showed insignificant difference in thiomersal activity. Low concentrations of propylene glycol and glycerol (2 to about 6.5%) significantly reduced the activity of thiomersal against P. aeruginosa. Higher concentrations up to about 40%, of the two cosolvents did not affect the antibacterial activity. Viscosity increasing agents significantly reduced the antibacterial activity of thiomersal. Low concentrations of 0.05% and 0.05-0.1% of ethylenediaminetetraacetic acid resulted in a significant decrease in thiomersal activity against S. aureus and P. aeruginosa , respectively. However, concentrations above 0.25 and 0.5 up to 4% caused significant increase on the antibacterial activity against the two later micro-organisms, respectively. Conclusions: Results showed that thiomersal formed complexes with ingredients containing polyhydroxy groups and chelating agents, thus thiomersal is not recommended to be used with such ingredients. Significance and Impact of the Study: This study highlighted the importance of proper selection of ingredients and preservatives for ophthalmic preparations.