Recent popularity of three-dimensional movies raised some concern about microbiological safety of glasses dispensed into movie theatres. In this study, we analysed the level of microbiological contamination on them before and after use and between theatres adopting manual and automatic sanitation systems. The manual sanitation system was more effective in reducing the total mesophilic count levels compared with the automatic system (P < 0.05), but no differences were found for coagulase-positive staphylococci levels (P = 0.22). No differences were found for mould and yeast between before and after levels (P = 0.21) and between sanitation systems (P = 0.44). We conclude that more evidences are needed to support microbiological risk evaluation.