This study aims to expand the knowledge about fecal contamination by humans and animals using Adenovirus (AdV) as bioindicators in different water sources from rural areas, to evaluate the viral infectivity, and to compare the different techniques used to detect the Human mastadenovirus (HAdV). For that, 124 samples were collected (86 from groundwater and 38 from surface water) along the Rio dos Sinos Basin. Escherichia coli count was carried out, and the samples were submitted for the detection and characterization tests of AdV by different methods (qPCR, multiplex qPCR, and nested PCR). In addition, the viral infectivity was realized by integrated cell culture quantitative PCR (ICC-qPCR). E. coli was detected in 63% of groundwater samples (geometric mean of 16.7 MPN/100 mL) and 68% (geometric mean: 5.08×102 MPN/100 mL) in surface waters. Among the viral indicator in the groundwater, the HAdV was detected in 49% of the samples, followed by Canine mastadenovirus (CAV, 20%), Bovine mastadenovirus (BAdV, 17%), Aviadenovirus (AvAdV, 15%), and Porcine mastadenovirus (PAdV, 03%). In surface water, HAdV was detected in 45%, followed by CAV (42%), BAdV (29%), and PAdV and AvAdV (13%). The quantification of genomic copies per liter ranged from 9.40×104 to 5.54×1010 gc/L. In groundwater samples, it was possible to observe infectious adenovirus in 12% of the samples, as well as in surface water for 18%. The results showed an increase in the sensitivity of positive samples when a combined set of techniques were used for HAdV detection.