Background Urban environments exert strong pressures on animal behavior, leading to altered fear responses to humans. Species with a longer history of urban presence and greater tolerance to urban environments are expected to show reduced fear responses towards humans. Here, we examined whether avian flight initiation distance (a proxy of fear)—the distance at which a bird flees from an approaching human—is associated with a species’ timing of urban colonization (i.e., when it has started to breed in urban areas) and with present-day urban tolerance (i.e., how common it is in the city). Unlike previous studies which paired avian fear responses and urbanization timing from different regions, we collected both in the same city (Prague, Czechia), minimizing regional differences in urban history and providing a more rigorous test of the link between urbanization timing and avian fear responses. Results Using standardized data from 4420 flight initiation distance observations across 68 species, we applied Bayesian phylogenetic mixed models while controlling for ecological and contextual variables. We found that species with a longer urban history (i.e., earlier timing of urban colonization) showed significantly shorter flight initiation distances, suggesting reduced fear responses. In contrast, present-day urban tolerance based on breeding commonness was not related to flight initiation distance variation. Conclusions We found that the timing of urban colonization better predicts reduced fear of humans in birds than present-day urban tolerance, emphasizing the role of long-term behavioral filtering and/or selection in shaping urban wildlife behavior. By explicitly separating urbanization time from contemporary urban commonness within a single city and analyzing individual-level fear responses, our study shows that earlier urban colonizers exhibit consistently shorter escape distances, reflecting cumulative long-term processes rather than short-term plasticity alone. These findings highlight the importance of incorporating urban colonization history into behavioral ecology and urban wildlife management frameworks.