The treatment of hospital effluents (HE) is a major concern, as they are suspected of disseminating drugs and antibiotic resistance determinants in the environment. In order to assess HE influence on wastewater treatment plant biomass, lab-scale conventional activated sludge systems (CAS) were continuously fed with real HE or urban effluent as a control. To gain insights into the main hurdles linked to HE treatment, we conducted a multiparameter study using classical physicochemical characterization, phase contrast and confocal laser scaning microscopy, and molecular biology (i.e., pyrosequencing) tools. HE caused erosion of floc structure and the production of extracellular polymeric substances attributed to the development of floc-forming bacteria. Adaptation of the sludge bacterial community to the HE characteristics, thus maintaining the purification performance of the biomass, was observed. Finally, the comparative metagenomic analysis of the CAS showed that HE treatment resulted in an increase of class 1 resistance integrons (RIs) and the introduction of Pseudomonas spp. into the bacterial community. HE treatment did not reduce the CAS process performance; nevertheless it increases the risk of dissemination into the environment of bacterial species and genetic determinants (RIs) involved in antibiotic resistance acquisition.