In a drinking water distribution system, biofilm-producing bacteria are considered an alarm bell for increased emergence of waterborne pathogens. This study aims to monitor the prevalence of biofilm-forming Aeromonas species in the drinking water distribution systems in different broiler chicken farms. The antimicrobial activity of thyme essential oil (TEO), thyme essential oil nano-emulsion (TEO-N), chitosan (CS), chitosan nanoparticles (CS-NPs), and both CS and CS-NP-based coating TEO against the different Aeromonas spp. was evaluated using the broth microdilution and agar well diffusion assay. The overall prevalence rate of Aeromonas spp. was 49.3% (74.0/150). The highest rate of Aeromonas isolates was noted in water drinkers and tanks 75.0% (30/40) and 62.5% (25/40), respectively) followed by feedstuff 40.0% (12/30). In contrast, the highest percentage of biofilm-producing Aeromonas spp. was Aeromonas hydrophila 70.0% (14/20) followed by Aeromonas caviae 30.0% (6/20). The fatal effect of CS-NPs against all isolated Aeromonas spp. was achieved 100% at 1.5 and 2.0 μg/mL. Moreover, chitosan nanoparticles coating thyme essential oil (CS-NPs/TEO) verified the lethal effect 100% on both A. hydrophila and A. caviae at the ratio of 1:1 and 1:0.75 μg/mL. In conclusion, the main source of Aeromonas spp. in the drinking water distribution system was the unhygienic status of water tanks and drinkers that allowed biofilm to produce due to aggregation of Aeromonas bacteria on the inner surface of that equipment. Both CS-NPs and CS-NPs/TEO could be applied as a sanitizer and/or disinfectant for Aeromonas biofilm control.