Conserving urban biodiversity is often promoted as a ‘win-win’ nature-based solution that can help align public health and biodiversity conservation agendas. Yet, research on the relationship between biodiversity and psychological well-being reveals inconsistent and complex results. This body of research is also restricted to a few socio-cultural and environmental contexts and tends to ignore differences in individual characteristics, such as nature relatedness (i.e., emotional affinity to nature) and ecological knowledge, that can influence people’s experience of biodiversity. The aim of this interdisciplinary research is to explore the relationships between biodiversity and psychological well-being, and test the moderating effect of nature relatedness and ecological knowledge on these relationships. An ecological survey was conducted in 24 small urban gardens in Israel to measure the richness and abundance of birds, butterflies and plants, as well as land cover characteristics. In parallel, a social survey (close-ended questionnaires) was conducted in-situ to measure psychological well-being, nature relatedness, ecological knowledge, perceived species richness and socio-demographic variables. Psychological well-being measures were mostly associated with the cover of woody species, perceived species richness, and to a lesser extent, with actual species richness and abundance, for all taxa. Nature relatedness moderated these relationships. Respondents with high nature relatedness demonstrated positive well-being-richness relationships, while those with intermediate, or low nature relatedness showed no, or even negative relationships, respectively. Opposite relationships were recorded for bird abundance. Overall, individuals demonstrated poor ecological knowledge and this variable moderated only few relations between well-being measures, perceived butterfly richness and bird abundance. Our results demonstrate that one-size-does-not-fit-all when considering the relationship between psychological well-being and biodiversity, and that affinity to nature is a key moderator for this relationship. Designing urban green spaces that provide inclusive and meaningful nature experiences and foster emotional affinity to nature, is therefore key to aligning ecological and social objectives for sustainable urban planning.