This study aims to assess microbiological contamination using a molecular tool for detection of multiple enteropathogens in a coastal ecosystem area in Rio de Janeiro, Brazil. Ten litres of superficial water samples were obtained during the spring ebb tide from sampling sites along the Jacarepagua watershed. Samples were concentrated using skimmed milk flocculation method for TaqMan array card (TAC), designed to identify 35 enteric pathogens simultaneously, and single TaqMan qPCR analysis for detecting human adenovirus (HAdV) and JC human polyomavirus (JCPyV) as faecal indicator viruses (FIV). TAC results identified 17 enteric pathogens including 4/5 viral species investigated, 8/15 bacteria, 4/6 protozoa and 1/7 helminths. Escherichia coli concentration was also measured as faecal indicator bacteria (FIB) using Colilert Quanti-Tray System with positivity in all samples studied. HAdV and JCPyV qPCR were detected in 8 and 4 samples, respectively, with concentration ranging from 8 × 102 to 2 × 106 genome copies/L. Partial nucleotide sequencing demonstrated the occurrence of species HAdV A, C, D, and F, present in faeces of individuals with enteric and non-enteric infections, and JCPyV type 3 (Af2), prevalent in a high genetically mixed population like the Brazilian. The diversity of enteropathogens detected by TAC emphasizes the utility of this methodology for quick assessment of microbiological contamination of the aquatic ecosystems, speeding up mitigation actions where the risk of the exposed population is detected, as well as pointing out the infrastructure gaps in areas where accelerated urban growth is observed.