It is important that operating room personnel monitor the correct amount of blood loss during surgery in order to properly replace lost volume. The aim of this study was to investigate the accuracy of operating room personnel in visually estimating blood loss in surgical sponges. We performed an observational study with comparative descriptive design at a university hospital including all members of the surgical team. In total, 163 observations were completed. The participants estimated the amount of blood in surgical sponges in 4 stations with varying amounts of blood and/or numbers of sponges. Data were analyzed using the Wilcoxon signed rank, Kruskal-Wallis, and Mann-Whitney tests. Both overestimations and underestimations occurred. Underestimations dominated and tended to increase with major amounts of blood. Operating room personnel miscalculated the amount of blood by a median value of 30% regardless of profession, years of experience, and self-assessed ability about visual estimation. This study highlights that assessments of patients’ conditions can be partially based on methods often demonstrated to be inaccurate. Inaccurate visual estimation of blood loss might endanger patient safety.