Assess healthcare workers' (HCW) attitudes toward universal masking, and gowns and gloves used as part of transmission-based precautions. Cross-sectional survey. Academic, tertiary care medical center in Baltimore, Maryland. HCW who work in patient care areas and have contact with patients. In May 2023, a 15-question web-based survey was distributed by the hospital's communications team via email. The survey contained questions to assess HCW perceptions of universal masking policies prior to the availability of COVID-19 vaccines and at the time of the survey, and the use of gowns and gloves for transmission-based precautions. Descriptive statistics were used to summarize data. Differences in agreement with universal masking over time, level of agreement with gown and glove policies, and with all PPE types across respondent characteristics were assessed. 257 eligible respondents completed the survey. Nurses and patient care technicians (43%) and providers (17%) were the most commonly reported roles. Agreement with universal mask use decreased from 84% early in the pandemic to 55% at the time of the survey. 70% and 72% of HCW agreed masks protect themselves and others, respectively. 63% expressed any level of annoyance with mask wearing, the most often due to communication challenges or physical discomfort. 75% agreed with gown use for antibiotic-resistant bacteria compared with 90% for glove use. The majority of HCW agree with the use of PPE to prevent pathogen transmission in the healthcare setting. Agreement with universal mask use for patient care shifted during the COVID-19 pandemic.