Animal facility personnel provide the husbandry and care of laboratory animals. We aimed to investigate their work-related quality of life, empathy and mental well-being. Participants living in Spain were contacted by email and asked to complete an anonymous online questionnaire, in which they answered the Professional Quality of Life scale, the Cognitive and affective empathy test, the Warwick–Edinburgh Mental Well-Being Scale, and their perceived human–animal interaction. Participants were asked whether they were receiving psychological therapy or were taking anxiolytics, hypnotics or antidepressant medication. The study comprised 80 participants. No differences were observed related to personal or professional variables. Participants working with small carnivores reported higher total empathy, and those working with non-human primates reported higher emotional comprehension. Higher human–animal interaction was reported by participants working with small carnivores, farm animals and non-human primates. More than half of the participants reported high levels of mental well-being, positively correlated with emotional comprehension, emphatic joy and compassion satisfaction. Participants working with farm animals reported higher levels of secondary traumatic stress that was positively correlated with human–animal interaction and negatively with mental well-being. Most participants reported low–average levels of burnout, which was negatively correlated with mental well-being. The percentage of animal facility personnel in psychotherapy was higher than in the general population, and the consumption of anxiolytics was a little lower and antidepressants higher. Overall, our results indicate that animal-facility personnel who felt stress or worse mental well-being were in therapy and took medication to improve their condition.