The main goal of this study was to apply magnetic bead surface functionalization in the form of immunomagnetic separation (IMS) combined with real-time polymerase chain reaction (qPCR) (IMS-qPCR) to detect Human mastadenovirus species C (HAdV-C) and F (HAdV-F) in water samples. The technique efficiency was compared to a nonfunctionalized method (ultracentrifugation) followed by laboratory detection. Tests were carried out to standardize IMS parameters followed by tests on 15 water samples concentrated by IMS and ultracentrifugation. Microscopic analyses detected a successful beads–antibody attachment. HAdV was detected up to dilutions of 10−6 by IMS-qPCR, and samples concentrated by IMS were able to infect cell cultures. In water samples, HAdV-C was detected in 60% (monoclonal) and 47% (polyclonal) by IMS-qPCR, while 13% of samples concentrated by ultracentrifugation gave a positive result. HAdV-F was positive in 27% of samples by IMS-qPCR (polyclonal) and ultracentrifugation and 20% by IMS-qPCR (monoclonal). The rate of detection varied from 4.55 × 102 to 5.83 × 106 genomic copies/L for IMS-qPCR and from 2.00 × 102 to 2.11 × 103 GC/L for ultracentrifugation. IMS showed to be a more effective concentration technique for HAdV than ultracentrifugation, improving the assessment of infectious HAdV in water resources.