Metagenomics has demonstrated its capability in outbreak investigations and pathogen surveillance and discovery. With high-throughput and effective bioinformatics, many disease-causing agents, as well as novel viruses of humans and animals, have been identified using metagenomic analysis. In this study, a VIDISCA metagenomics workflow was used to identify potential unknown viruses in 33 fecal samples from asymptomatic long-tailed macaques (Macaca fascicularis) in Ratchaburi Province, Thailand. Putatively novel astroviruses, enteroviruses, and adenoviruses were detected and confirmed by PCR analysis of long-tailed macaque fecal samples collected from areas in four provinces, Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan, where humans and monkeys live in proximity (totaln= 187). Astroviruses, enteroviruses, and adenoviruses were present in 3.2%, 7.5%, and 4.8% of macaque fecal samples, respectively. One adenovirus, named AdV-RBR-6-3, was successfully isolated in human cell culture. Whole-genome analysis suggested that it is a new member of the speciesHuman adenovirus G, closely related to Rhesus adenovirus 53, with evidence of genetic recombination and variation in the hexon, fiber, and CR1 genes. Sero-surveillance showed neutralizing antibodies against AdV-RBR-6-3 in 2.9% and 11.2% of monkeys and humans, respectively, suggesting cross-species infection of monkeys and humans. Overall, we reported the use of metagenomics to screen for possible new viruses, as well as the isolation and molecular and serological characterization of the new adenovirus with cross-species transmission potential. The findings emphasize that zoonotic surveillance is important and should be continued, especially in areas where humans and animals interact, to predict and prevent the threat of emerging zoonotic pathogens.