Simple Summary Animal welfare is a current societal concern, and non-invasive indicators are required to assess the welfare state of animals. The selection of horses for certain functions and individual differences could result in different strategies to deal with stressors. This is why in the present study we assessed behavioral and physiological responses of two types of horses (working horses and Chilean rodeo horses) to a handling test (bridge test). We evaluated five behaviors, the number of attempts, and the time required to cross a bridge. Heart rate and the variability of heart rate were registered with a polar system during rest and during the bridge test. Chilean rodeo horses displayed several active behaviors in order to avoid the bridge and required a higher number of attempts to complete the task, but physiologically responded better. On the other hand, all working horses crossed the bridge on the first attempt, without refusal behaviors, but physiologically did not respond as well as Chilean rodeo horses. Behavior does not always correlate with physiological data, and needs to be interpreted carefully when assessing horse welfare. Non-invasive measures are preferred when assessing animal welfare. Differences in behavioral and physiological responses toward a stressor could be the result of the selection of horses for specific uses. Behavioral and physiological responses of working and Chilean rodeo horses subjected to a handling test were assessed. Five behaviors, number of attempts, and the time to cross a bridge were video recorded and analyzed with the Observer XT software. Heart rate (HR) and heart rate variability (HRV), to assess the physiological response to the novel stimulus, were registered with a Polar Equine V800 heart rate monitor system during rest and the bridge test. Heart rate variability data were obtained with the Kubios software. Differences between working and Chilean rodeo horses were assessed, and within-group differences between rest and the test were also analyzed. Chilean rodeo horses presented more proactive behaviors and required significantly more attempts to cross the bridge than working horses. Physiologically, Chilean rodeo horses presented lower variability of the heart rate than working horses.