Background Few studies have explored the microbial contamination of glove boxes in clinical settings. The objective of this observational study was to investigate whether a new glove packaging system in which single gloves are dispensed vertically, cuff end first, has lower levels of contamination on the gloves and on the surface around the box aperture compared with conventional glove boxes. Methods Seven participating sites were provided with vertical glove dispensing systems (modified boxes) and conventional boxes. Before opening glove boxes, the surface around the aperture was sampled microbiologically to establish baseline levels of superficial contamination. Once the glove boxes were opened, the first pair of gloves in each box was sampled for viable bacteria. Thereafter, testing sites were visited on a weekly basis over a period of six weeks and the same microbiological assessments were made. Results The surface near the aperture of the modified boxes became significantly less contaminated over time compared with the conventional boxes (P<0.001), with an average of 46.7% less contamination around the aperture. Overall, gloves from modified boxes showed significantly less colony-forming unit contamination than gloves from conventional boxes (P<0.001). Comparing all sites over the entire six-week period, gloves from modified boxes had 88.9% less bacterial contamination. Conclusion This simple improvement to glove box design reduces contamination of unused gloves. Such modifications could decrease the risk of microbial cross-transmission in settings that use gloves. However, such advantages do not substitute for strict hand hygiene compliance and appropriate use of non-sterile, single-use gloves.