Guinea pigs are included in various animal-assisted interventions (AAIs), but no research has been published to date on behavioral changes in guinea pigs interacting with humans. The goal of this study was to evaluate the behavior in guinea pigs during animal-assisted therapy (AAT) and to identify factors that influence their stress and well-being. Five guinea pigs were studied during 50 observations in a randomized controlled within-subject design with repeated measurement. All guinea pigs were tested under all the following conditions: (1) therapy setting with retreat possibility (n = 20), (2) therapy setting without retreat possibility (n = 10), and (3) control setting without human interaction (n = 20). Behavior was coded according to a specifically designed ethogram using continuous recording and focal animal sampling with The Observer® XT 12.5. The data were analyzed using generalized linear mixed models with SPSS®, version 22.0. Results show that the frequency but not the duration of hiding was significantly increased in the therapy setting with retreat possibility compared to the control condition. During therapy with retreat possibility, the number of comfort behavior episodes stayed constant, while the number of startling and explorative behavior and the duration of locomotion increased significantly in comparison to the control setting. During therapy without retreat possibility, the frequency of freezing was increased significantly in comparison to the therapy setting with retreat possibility and the control setting. Comfort behavior was never observed during therapy without retreat possibility. This study provides evidence that the possibility of retreat is instrumental in reducing stress and should be provided during AAT using guinea pigs. In this form, AAT elicits limited stress and may possibly even provide enrichment. Further research is needed to understand factors influencing guinea pig behavior to ensure animal welfare in AAIs in the future.