To mitigate the effects of anthropogenic environmental impacts, conservation education (CE) aims to increase public awareness of biodiversity and encourage the uptake of pro-environmental behaviours (PEB). One way of achieving this goal is the use of controlled animal interactions with animal ambassadors to promote CE goals. This study aimed to determine how differing experiences with ambassador Humboldt penguins (Spheniscus humboldti) affected visitors’ knowledge of conservation actions (CA) and their likelihood to perform PEBs in a UK zoo. A quasi-experimental repeated measures questionnaire was used to investigate this aim. Visit types were categorised as (a) ‘penguin parade’, (b) ‘animal encounter’, (c) ‘exhibit viewing’ and (d) a ‘control’ groups. A Kruskal–Wallis test was used to determine if responses to rating scale items related to PEBs differed according to experience. No significant (p > 0.05) differences were detectable between groups. Qualitative data provided in open ended questions relating to CAs were coded into themes and the percentage of responses were calculated for each theme. Whilst the ‘exhibit viewing’ group mentioned sustainable fish significantly more (p = 0.017) than the ‘control’ group, no significant difference was found for any other pair-wise comparison (p > 0.05 in all cases). Overall, this study highlights that zoo visitors gained specific conservation-based knowledge and had higher behavioural intentions towards PEBs than they previously had. However, no evidence was found to suggest that animal encounters improved CE outcomes. To ensure best practice is followed and CE outcomes are considered equally alongside ambassador animal welfare concerns, further research is recommended.