Bats have historically interacted in different ways with human societies. These interactions have been expressed in the cultural manifestations and cognitive and affective links that we have towards this type of organisms, which arise from perceived and real beliefs, knowledge and impacts that include them, and generate affection, attachment, relevance, aversion and wrong practices on these species and their habitats in urban and rural settings. In this study, I examined for the first time in an urban-rural gradient, how attitudes and biophilic values vary and their impact on human-bat interactions. A Bat Attitude Questionnaire (BAQ) was carried out on 210 men and women in urban, peri-urban and rural areas in and around of the Cali city, Colombia. Positive, negative and neutral attitudes and nine biophilic values towards this group were evaluated. The results showed that a higher level of education generated greater ecological-scientific value and more positive attitudes towards these organisms in all areas. However, ecological knowledge of the eating habits of this order is not always associated as a possible benefit. The dominionistic and negativistic values were high due to the low tolerance for sharing habitats as well as the fear caused by the association of bats with diseases, causing several forms of exclusion and a variety of reasons to kill a bat. Humanistic and moralistic values were also expressed by a high regard for them. The lack of aesthetic beauty of this group was recognized by most respondents. Human-bats interactions varied throughout the urban-rural gradient, with the rural area being the one with the highest number of registered interactions. The biophilic values did not show significant variations throughout the gradient. There were a greater number of positive than negative attitudes and a high willingness to participate in bat conservation projects. Belief in myths, although it was widespread in all areas and distributed similarly among young people and adults, did not seem to prevail among respondents and did not generate intolerance about these organisms. Additionally, the findings indicated that gender and age do not influence attitudes or biophilic values. These results significantly expose that the human-bat relationship is complex and can be the result of multiple social and cultural factors that must be understood and addressed in the development of actions and instruments that promote the conservation of this group of organisms.