Healthcare personnel (HCP) frequently wear gloves when they care for patients in Standard Precautions to prevent contact with potentially infectious blood or body fluids. When HCP use gloves appropriately they reduce the risk of cross-contamination and decrease the risk of healthcare-associated infections (HAI). However, if HCP use gloves inappropriately they may inadvertently spread pathogens to patients and the patients’ environment. This study used a descriptive structured observational design to investigate three aspects of HCP glove use in a United States long-term care facility (LTCF). First, the PI examined the degree of inappropriate HCP glove use in a random sample of 76 HCP. Results indicate that the HCP used gloves inappropriately, failing to change gloves 66% of the time when a glove change was indicated. Over 44% of the HCP gloved touch points were defined as contaminated. Second, the PI examined the reliability of a new glove use tool (GUST). Results indicate the GUST is a reliable tool when used by trained observers documenting HCP glove use during toileting and perineal care events in LTCF, with intraclass correlation coefficients (ICC 2,1) over 0.75 for indicators of inappropriate glove use. Third, exploratory analysis indicated significant differences between inappropriate glove use in females and males. Female HCP had significantly more failed glove changes and contaminated touch points than male HCP in this study (p = 0.003). Future research studies should assess US HCP glove use to provide data needed for development of strategies to improve HCP glove use and reduce HAI.