Purpose <p>To investigate the effectiveness of preservative-free multi-dose eye drop technology in preventing contamination.</p> Study design <p>Prospective study</p> Methods <p>In this study, the caps, first and second drops of preservative-free multidose brimonidine (PF-MDB) and multidose brimonidine with purite preservative (P-MDB) used by 54 glaucoma patients were analysed on both chocolate and sheep blood agar. The patients used the drops at 3-week intervals. The contents of the eye droppers were examined for contamination by piercing the bottom of the eye dropper in accordance with aseptic techniques.</p> Results <p>The contamination levels detected using chocolate agar in the caps and the first drops of PF-MDBs were significantly higher than in P-MDBs (<i>p</i>=0.006 and p&lt;0.001, respectively). The contamination levels detected using blood sheep agar in the caps and the first and second drops of PF-MDBs were significantly higher than in P-MDBs (<i>p</i>=0.001, ‹0.001, 0.006, respectively). No growth was found inside the eye dropper bottles of either PF-MDB or P-MDB drops. There was a significantly higher level of contamination in the first drops of PF-MDB compared to the second drops (p&lt;0.001).</p> Conclusion <p>PF-MDB bottle technology effectively prevents microbial contamination in the bottle without preservatives. However, due to the lack of a preservative, there is increased bacterial and fungal growth associated with contamination on the cap, as well as in the first and second drops, compared to P-MDB.</p>