This study aims to isolate and identify both diseased and healthy fish pathogens of Ctenopharyngodon idella, Labeo rohita and Oreochromis niloticus and assess their antibacterial and biofilm supressing activities against fish pathogens. It explores their potential to inhibit and degrade biofilms, serving as an alternative to antibiotics in aquaculture while enhancing fish health and disease resistance. Furthermore, the research endeavors to assess the biofilm degradation potential of antibiotics and probiotics, both individually and in combination. The biofilm-forming potential of pathogens was assessed both qualitatively and quantitatively using the Congo red assay, cover slip, and test tube methods. Additionally, genomic sequencing through 16S rRNA ribotyping revealed the species level identification of four pathogenic and twelve probiotic strains. Three pathogens, Staphylococcus sciuri, Pseudomonas aeruginosa, and Staphylococcus xylosus, showed significant biofilm formation at day 5, while the pathogen Niallia circulans expressed maximum biofilm formation on day 7. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics were evaluated against pathogenic strains. Antibiotic susceptibility testing revealed significant inhibition zones. MIC and MBC values ranged from 0.10 mg/ml to 85.00 mg/ml, with the agar well and disk diffusion methods demonstrating strong inhibitory effects against the pathogenic strains. Notably, fish probiotics either alone or in combination with antibiotics exhibited significant inhibition and anti-biofouling activity across three different concentrations (1/2 MIC, 1MIC, 2XMIC). The biofilm eradication values were statistically significant (p <
0.005). The findings affirm the effectiveness of the antibiotics (ampicillin, levofloxacin, kanamycin and oxytetracycline) and probiotics (Bacullus altitudinis, Bacillus pumilus, Mammaliicoccus sciuri) employed in preventing and dispersing biofilms formed by isolated fish pathogens (S. sciuri, P. aeruginosa and N. circulans). The current study explores the use of probiotics to enhance fish immunity, reduce disease risk without promoting antibiotic resistance, and disrupt pathogenic biofilms to control infections. Unlike antibiotics, probiotics are biodegradable and eco-friendly, minimizing harm to aquatic ecosystems and beneficial microbes.