Background/aim: Povidone-iodine (PI, Betadine) is routinely used as a preoperative topical antiseptic in cataract surgery as it has been shown to reduce the incidence of postoperative endophthalmitis. However, the concentration used clinically is variable. In vitro studies have shown that PI is paradoxically more effective at lower concentration. This study was undertaken to determine if this effect was reproducible in vivo. Methods: A prospective randomised double blind study was carried out in the ophthalmic theatre in a district general hospital. 105 patients attending for routine cataract surgery were randomly allocated to have their conjunctival fornices irrigated preoperatively with either PI 1% (group A) or PI 5% (group B). Conjunctival swabs were taken, in identical fashion, both before and 1 minute after irrigation. The number and species of bacterial colonies cultured from each swab was counted. The difference in the median number of bacterial colonies from pre-irrigation to post-irrigation cultures was then compared between the groups. Results: Bacterial cultures were gained from 100 patients (33 male, 67 female, mean age 74 years, range 30-95 years). Group B (5% PI) showed a decrease in median colony forming units (CFU) preirrigation from 100 to 40 CFU post-irrigation (a drop of 60%). This was greater than in group A (1% PI) where the reduction was 120 CFU pre-irrigation to 100 CFU post-irrigation (a drop of 16.7%) (Mann-Whitney test, p<0.05). At higher initial bacterial loads (CFU pre-irrigation >1000), the difference in median between the two groups became larger as the number of pre-irrigation bacteria increased. In group B pre-irrigation CFU reduced from 3340 to 110 post-irrigation (a drop of 96.7%) compared with group A: 5000 CFU pre-irrigation to 3000 post-irrigation (a drop of 40%) (Mann-Whitney test, p=0.0014). Conclusion: Despite in vitro evidence of higher bactericidal efficacy of PI at more dilute concentrations, 5% PI is more effective than 1% PI in decreasing the human conjunctival bacterial flora in vivo, particularly in the presence of heavier initial bacterial load.