Use of gloves in dentistry is primarily for protection of the healthcare worker, yet little information is available regarding potential patient issues such as microbial contamination of gloves before and during use. The purposes of this study were to compare gloves from newly opened boxes with those from boxes that had been in dental operatories until they were nearly empty and to determine if gloves are contaminated though diagnostic procedures and rubber dam placement. Eight endodontic residents provided samples by streaking gloved fingers on nutrient agar plates. Results showed no statistically significant difference between counts from new boxes versus nearly empty boxes (n = 32, p < 0.6216). Gloves after rubber dam placement yielded a mean colony count of 158 versus a 1.5 mean of fresh gloves (n = 64, p < 0.0001). These results suggest that the use of new gloves before opening a tooth for endodontic therapy may be warranted.