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Purpose: Certain ocular resident or pathogenic microbes may remain viable in the presence of multi-purpose disinfectant solutions (MPDSs), subsequently developing biofilms inside contact lens storage cases (CLSCs) which pose a risk of infection to wearers. This study evaluated the formation of ocular microbiota biofilms exposed to three top selling MPDS., Methods: Crystal violet assay was carried out for the verification of biofilm formation. The in vitro assays evaluated Pseudomonas aeruginosa UFPEDA 416 and Staphylococcus aureus UFPEDA 02 exposure of 48 h to MPDS, as well as the use of 40 KHz ultrasound at the beginning and with 24 h immersion in the MPDS. Subsequently, in vivo assays evaluated the formation of microbial biofilms on the CLSC walls containing silicone-hydrogel contact lenses immersed in MPDS from 15 healthy volunteer patients, who had been wearing the lenses for 7 days., Results: Biofilms were inhibited by 26%-98% in the in vitro assays, with a statistically significant difference only for P. aeruginosa UFPEDA 416 exposed to diluted MPDS. Most inhibitions occurred moderately and weakly. In addition, adherent cells were detected in more than 90% of the tests. Biofilm was not inhibited in more than one third of the results, nor was it disturbed, especially with the ultrasound treatments. The average of obtained optical densities at 590 nm was between 0.6 and 0.8 in the in vivo assays. The results were similar between the CLSC right and left wells. There was a correlation between microbial biofilm formation and the type of MPDS tested, with statistical difference between the three treatments., Conclusion: MPDS promoted a partial inhibition of microbial biofilm formation but only one MPDS proved to be more effective in vitro and in vivo . This study, however, could not distinguish the effect of possible errors in the good hygiene practices of the users., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Middle East African Journal of Ophthalmology.)