While playing a critical role in preventing healthcare-associated infections, patient room cleaning is often unsatisfactorily performed. To improve patient room cleaning, a human factors and systems engineering (HFSE) approach is needed to understand the complex cleaning process and associated work system factors.We conducted an observational study to assess the performance of environmental care (EVC) associates during daily patient room cleaning and identify work system factors influencing their performance.This study was conducted in eight adult medicine inpatient units at a large urban academic medical center. An HFSE researcher shadowed 10 day-shift EVC associates performing daily patient room cleanings and used a semi-structured observation form to collect quantitative data (e.g., duration of room cleaning, orders for surface cleaning) and qualitative data (e.g., challenges to patient room cleaning). Descriptive statistics (e.g., median, interquartile range) were reported for cleaning performance, and bivariate and regression analyses were conducted to identify factors influencing cleaning performance. We also performed link analyses of the workflow of EVC associates and qualitative analyses of observer notes to identify challenges to daily patient room cleaning.We observed 89 patient room cleanings. Median duration of cleaning a room was 14 minutes, and median percentage of surfaces cleaned in a room was 63%. High-touch surfaces that were frequently missed during daily cleaning included the bedrails, telephone, patient and visitor chairs, and cabinet. Work system factors that could influence cleaning performance included the type of unit, the presence of the patient and family members in the room, cleaning patterns and orders of EVC associates, and interruptions EVC associates encountered while cleaning.Daily patient room cleaning was influenced by a number of work system factors. To improve daily patient room cleaning, multifaceted interventions are needed to address these system-level factors.