Outbreaks of human illness have been linked to visiting settings with animal contact throughout developed countries. This study details an observational study of hand hygiene tool availability and recommendations; frequency of risky behaviour; and handwashing attempts by visitors in Kansas (9) and Missouri (4), USA, petting zoos. Handwashing signs and hand hygiene stations were available at the exit of animal-contact areas in 10/13 and 8/13 petting zoos, respectively. Risky behaviours were observed being performed at all petting zoos by at least one visitor. Frequently observed behaviours were as follows: children (10/13 petting zoos) and adults (9/13 petting zoos) touching hands to face within animal-contact areas; animals licking children’s and adults’ hands (7/13 and 4/13 petting zoos, respectively); and children and adults drinking within animal-contact areas (5/13 petting zoos each). Of 574 visitors observed for hand hygiene when exiting animal-contact areas, 37% (n = 214) of individuals attempted some type of hand hygiene, with male adults, female adults and children attempting at similar rates (32%, 40% and 37%, respectively). Visitors were 4.8× more likely to wash their hands when a staff member was present within or at the exit to the animal-contact area (136/231, 59%) than when no staff member was present (78/343, 23%; P < 0.001, OR = 4.863, 95% CI = 3.380–6.998). Visitors at zoos with a fence as a partial barrier to human–animal contact were 2.3× more likely to wash their hands (188/460, 40.9%) than visitors allowed to enter the animals’ yard for contact (26/114, 22.8%; P < 0.001, OR = 2.339, 95% CI = 1.454–3.763). Inconsistencies existed in tool availability, signage and supervision of animal contact. Risk communication was poor, with few petting zoos outlining risks associated with animal contact, or providing recommendations for precautions to be taken to reduce these risks.