It is becoming increasingly evident that cities are important places for biodiversity. Biodiverse urban forests are vital green areas within cities and have favorable impacts on the citizens, including their health. We focused on the effect of the urban forest environment on biodiversity in Prague, the capital of the Czech Republic. We used a multi-taxon approach with five taxa of different ecological demands: butterflies, bees and wasps, vascular plants, mosses, and lichens. We modeled their responses to the various urban forest attributes at four hierarchical levels – plot, permeability, forest, and landscape. Our results revealed that temporally continuous forests dominated by native oaks with open canopies, a high number of admixed and interspersed tree species and shrubs, together with scattered trees in the surrounding landscape, were optimal biodiverse forest environments. The most influential parameter that positively influenced bees and wasps, plants, and lichens at the plot level was canopy openness. We found that the permeability was suitable mainly on 20 m surroundings and increasing coverage of native oaks and tree species richness were the most important parameters. Continuity was the only found parameter that influenced mosses at the forest level. Scattered tree vegetation was the most important landscape parameter and positively drove the species richness of bees and wasps. Forest management methods can relatively easily solve the improvement of the scattered light gap structure within urban forests. Applying traditional forest management (pasture management, controlled burning and/or coppicing) is also an option but requires sensitive communication with the public. The canopy cover has been used as an indicator of urban forest health conditions, now indicating that artificial disturbances could be important issues for urban forest management and planning in the future. Therefore, active forest management is an essential method for biodiversity maintenance. We conclude that urban forests have a high potential for increasing native biodiversity. The response of the studied groups in urban forests was complementary. The resulting biodiverse stages of urban forests are akin to the established idea of the open temperate deciduous woodlands.