The occurrence of Aeromonas spp. within biofilms formed on stainless steel (SS), unplasticized polyvinyl chloride (uPVC) and glass (GL) substrata was investigated in modified Robbins Devices (MRD) in potable (MRD-p) and recycled (MRD-r) water systems, a Biofilm Reactor™ (BR) and a laboratory-scale pipe loop (PL) receiving simulated recycled wastewater. No aeromonads were isolated from the MRD-p whereas 3–10% of SS and uPVC coupons (mean 3.85 CFU cm−2 and 12.8 CFU cm−2, respectively) were aeromonad-positive in the MRD-r. Aeromonads were isolated from six SS coupons (67%) (mean 63.4 CFU cm−2) and nine uPVC coupons (100%) (mean 6.50×102 CFU cm−2) in the BR™ fed with recycled water and from all coupons (100%) in the simulated recycled water system (PL). Mean numbers of aeromonads on GL and SS coupons were 5.83×102 CFU cm−2 and 8.73×102 CFU cm−2, respectively. No isolate was of known human health significance (i.e. Aeromonas caviae, A. hydrophila or A. veronii), though they were confirmed as Aeromonas spp. by PCR and fluorescence in situ hybridization (FISH). Challenging the PL biofilms with a slug dose of A. hydrophila (ATCC 14715) showed that biofilm in the PL accumulated in the order of 103–104A. hydrophila cm−2, the number of which decreased over time, though could not be explained in terms of conventional 1st order decay kinetics. A sub-population of FISH-positive A. hydrophila became established within the biofilm, thereby demonstrating their ability to incorporate and persist in biofilms formed within distribution pipe systems. A similar observation was not made for culturable aeromonads, though the exact human health significance of this remains unknown. These findings, however, further question the adequacy of culture-based techniques and their often anomalous discrepancy with direct techniques for the enumeration of bacterial pathogens in environmental samples.