Background: The probiotics’ auto-aggregation and biofilm formation abilities have a significant role in the development of biotechnological processes.Objective: The aim of this study was to evaluate the biofilm formation and auto-aggregation abilities of novel, targeted aqua-probiotics isolated from aquatic organisms.Methods: The biofilm formation abilities of Lactobacillus delbrueckii str. UZ-1, Lactiplantibacillus plantarum str. R3, Lactococcus str. UZ-2, Enterococcus faecium str. R2, Pediococcus acidilactici str. N from the culture collection of the Microbiology of the Academy of Sciences of the Republic of Uzbekistan, Bacillus subtilis str. 1R, Bacillus amyloliquefaciens str. 4R and from the culture collection of the Southern Federal University of Russa and Lacticaseibacillus rhamnosus str. 1A and Enterococcus str. 9-3 from the culture collection of the Armenian National Agrarian University were assessed.Results: According to the investigations, the biofilm formation abilities of Lactobacillus delbrueckii str. UZ-1, Lactiplantibacillus plantarum str. R3, Lactococcus str. UZ-2, Enterococcus faecium str. R2, Pediococcus acidilactici str. N, Bacillus subtilis str. 1R, Bacillus amyloliquefaciens str. 4R, Bacillus amyloliquefaciens str. 5R, Lacticaseibacillus rhamnosus str. 1A and Enterococcus str. 9-3 were 0.119 ± 0.05D, 0.113 ± 0.065D, 0.196 ± 0.04D, 0.116 ± 0.01D, 0.152 ± 0.05D, 0.74 ± 0.15D, 2.621± 0.55D, 1.831 ± 0.45D, and 0.227 ± 0.04D and 0.483 ± 0.15D respectively. The highest rate of auto-aggregation was shown by Bacillus amyloliquefaciens str. 5R, and Bacillus amyloliquefaciens str. 4R was the strain with the highest ability to form biofilm. These two Bacillus strains are also distinguished by the highest DNA protective properties and relatively low antioxidant activity. Despite the fact that Bacillus amyloliquefaciens str. 5R showed the highest rate of auto-aggregation after 2 hours, this strain showed the lowest level of auto-aggregation among the studied strains after 24 hours. The Enterococcus str. 9-3 strain with the highest antioxidant activity showed 0.483 ± 0.15D biofilm formation ability.Conclusion: The novel targeted aquaprobiotics have distinct biofilm formation and aggregation properties, which are important to consider when planning appropriate biotechnological processes, requiring specific membrane properties of probiotics.Graphical Abstract: Membrane properties of novel targeted aquaprobiotics.Keywords: Lactobacilli, aqua-probiotic, antioxidant activity, biofilm formation, aggregation, Enterococcus str. 9-3