Background: Hand hygiene is key to preventing health care-associated infections. Human observation is the gold standard for measuring compliance, but its utility is increasingly being questioned with calls for the use of video monitoring approaches. The utility of video-based systems to measure compliance according to the WHO 5 moments is largely unexamined, as is its acceptability amongst health care workers (HCW) and patients. This study examined HCW acceptability of video monitoring for hand hygiene auditing. Methods: Following trial of a video monitoring system (reported elsewhere), 5 participating HCW attended 2 in-depth group interviews where they reviewed the footage and explored responses to the approach. Transcripts were analyzed using thematic analysis. Results: Four themes were identified: 1) Fears; 2) Concerns for patients; 3) Changes to feedback; and 4) Behavioral responses to the cameras. HCWs expressed fears of punitive consequences, data security, and confidentiality. For patients, HCWs raised issues regarding invasion of privacy, ethics, and consent. HCWs suggested that video systems may result in less immediate feedback but also identified potential to use the footage for feedback. They also suggested that the Hawthorne Effect was less potent with video systems than human observation. Conclusions: The acceptability of video monitoring systems for hand hygiene compliance is complex and has the potential to complicate practical implementation. Additionally, exploration of the acceptability to patients is warranted. Checklist: COREQ