Urban environments may negatively affect the development of organisms. In host-pathogen/parasite systems, this impact may lead to increased manifestations of pathogens that decrease the success of their hosts in urban environments compared to rural ones. We tested this hypothesis in the solitary bee Osmia bicornis L. We estimated the development of bees, their reproductive success and the manifestation of different pathogens and nest parasites along an urbanisation gradient. We conducted the experiment in an urbanisation gradient in sites representing three environments: urban, suburban and rural. First, we analysed the manifestation of bacterial and fungal microorganisms in pollen loads, within dried/mummified individuals, on the surface of cocoons and on the surface of diapausing adult individuals by using the MALDI-TOF MS technique. We also verified genetic samples from diapausing individuals for the presence of the parasitic Nosema apis (Zandler, 1909) and N. ceranae (Fries et al., 1996) species. Finally, we assessed the level of reproductive success and manifestations of brood parasites. Not any biological material from the nests was infected by pathogenic microorganisms. This result indicates that the nests are not a reservoir of the pathogenic bacteria and that O. bicornis offspring are not a source or vectors of these pathogens to the surrounding environment and indirectly to other bee species. In urban sites, there was a lower number of parasites than in suburban and rural environments. The presence of parasites was negatively correlated with the reproductive success and may be a limiting factor for O. bicornis populations. We also found that urban sites had the highest indices of reproductive success and the lowest number of breeding failures compared to suburban and rural sites. Moreover, bacterial and fungal transmission is not a serious threat in the studied region.