The present study aimed to identify the antibacterial bioactive compounds in Withania roots and Basella leaves against Aeromonas infection in Nile tilapia. Withania ethanol extract (EW, ZoI 10.72 ± 1.028 mm) and Basella methanol extract (MB, ZoI 10.83 ± 1.058 mm) showed the highest antibacterial activity in disc diffusion assay.In in vivo experiment, Nile tilapia (25.61 ± 0.31 g) were fed basal, EW and MB (both at 0.7 g/kg feed) diets for 15 days either before being intraperitoneally injected with Aeromonas and basal diet feeding for 7 days (prophylactic) or after bacterial injection and basal diet feeding for 7 days (therapeutic). Fish fed EW and MB fortified diets showed significantly (p < 0.05) lower mortality, alkaline phosphatase, glutamic oxaloacetic transaminase, glutamic pyruvate transaminase activities, HSP70, Caspase3, IFN-ɣ, IL-1 β, TNFα level, while significantly (p < 0.05) higher weight gain, protein efficiency ratio, respiratory burst, lysozyme, phagocytotic activity, plasma total protein, total immunoglobulin content, RBC, WBC count, haemoglobin, haematocrit, mean corpuscular haemoglobin concentration, mean corpuscular volume, lymphocytes, neutrophils, monocytes percentages and superoxide dismutase, catalase, glutathione peroxidase activities compared to basal diet-fed fish. EW showed significantly (p < 0.05) better antibacterial efficacy compared to MB. Prophylactic treatment showed better results compared to therapeutic treatment. Bioassay-guided chromatography and GC-MS analysis revealed the presence of hexadecanoic acid, phytol, cis, cis, cis-7,10,13-hexadecatrienal in EW and α-tocopherol, 1-(+)-ascorbic acid 2,6-dihexadecanoate, 1-heptatriacotanol in MB, which was reported to have antibacterial, antioxidative and immunostimulatory potential. Present results indicate prospective use of EW as a prophylactic immunostimulant and effective therapeutic remedy against Aeromonas in Nile tilapia.