Introduction: A decline in direct experience with nature can lead to disaffection of natural environments, wildlife, and public indifference towards biodiversity conservation. This study measured on affective attitude towards wildlife (i.e., preferences for and willingness to coexist with 22 animal species) and examined the relationships between these attitudes and childhood experiences with nature. Methods: A face to face interview was carried out in rapidly urbanizing Malaysia for both urban and suburban 357 adults (age > 20 years old). Results: It found that Malaysian people liked several insects and squirrels, but disliked mammals generally. Mediation analysis, with controlling sociodemographic factors (gender, age and ethnicity), showed that childhood nature experience was positively associated with preference for wild animals (standardized path coefficient = 0.18; p < 0.001), and the preference had a strong correlation with willingness to coexistence (standardized path coefficient = 0.61; p < 0.001) with the animals. Childhood nature experience, however, had limited effects on willingness to coexist with the animals via the preference, particularly for unfavourable animals. These results suggest that preference and willingness scores, even though they were significantly correlated, were different sides of affective attitudes toward animals. Conclusions: Therefore, to promote biodiversity conservation programs, we need strategies to increase acceptance of wild animals via relevant environmental education and public communication, as well as opportunities for nature activities for children.