The evaluation of effluent wastewater quality mainly relies on the assessment of conventional bacterial indicators, such as fecal coliforms and enterococci; however, little is known about opportunistic pathogens, which can resist chlorination and may be transmitted in aquatic environments. In contrast to conventional microbiological methods, high-throughput molecular techniques can provide an accurate evaluation of effluent quality, although a limited number of studies have been performed in this direction. In this work, high-throughput amplicon sequencing was employed to assess the effectiveness of chlorination as a disinfection method for secondary effluents. Common inhabitants of the intestinal tract, such as Bacteroides, Arcobacter and Clostridium, and activated sludge denitrifiers capable of forming biofilms, such as Acidovorax, Pseudomonas and Thauera, were identified in the chlorinated effluent. Chloroflexi with dechlorination capability and the bacteria involved in enhanced biological phosphorus removal, i.e., Candidatus Accumulibacter and Candidatus Competibacter, were also found to resist chlorination. No detection of Escherichia indicates the lack of fecal coliform contamination. Mycobacterium spp. were absent in the chlorinated effluent, whereas toxin-producing cyanobacteria of the genera Anabaena and Microcystis were identified in low abundances. Chlorination significantly affected the filamentous bacteria Nocardioides and Gordonia, whereas Zoogloea proliferated in the disinfected effluent. Moreover, perchlorate/chlorate- and organochlorine-reducing bacteria resisted chlorination.