Petting zoos are popular attractions, but can also be associated with zoonotic disease outbreaks. Hand hygiene is critical to reducing disease risks; however, compliance can be poor. Video observation of petting zoo visitors was used to assess animal and environmental contact and hand hygiene compliance. Compliance was also compared over five hand hygiene intervention periods. Descriptive statistics and multivariable logistic regression were used for analysis. Overall hand hygiene compliance was 58% (340/583). Two interventions had a significant positive association with hand hygiene compliance [improved signage with offering hand sanitizer, odds ratio (OR) 3·38, P<0·001; verbal hand hygiene reminders, OR 1·73, P=0·037]. There is clearly a need to improve hand hygiene compliance at this and other animal exhibits. This preliminary study was the first to demonstrate a positive impact of a hand hygiene intervention at a petting zoo. The findings suggest that active, rather than passive, interventions are more effective for increasing compliance.