The development of resistance to antimicrobial agents has been the major concern in the aquaculture industry. Essential oil nanoemulsions can be an effective alternative to the synthetic antimicrobial compounds, as they are efficient and safer to the environment. This study deals with the formulation, characterisation, application and biosafety of the black pepper oil nanoemulsion prepared using high-pressure homogenization method. A stable formulation of black pepper oil nanoemulsion was obtained with the oil and surfactant (Tween 80) ratio of 1:2. The mean droplet size and polydispersity index of the nanoemulsion was found to be 30.3 ± 2 nm and 0.21 respectively. The antibacterial activity of the black pepper oil nanoemulsion against the bacterial pathogen Pseudomonas aeruginosa as studied by microbroth dilution method was found to be 0.234 μl/ml. SEM analysis of the nanoemulsion treated bacterial cells showed significant membrane damage and the results were corroborative with the cell membrane integrity study. The MIC of black pepper oil nanoemulsion was found to be very effective in inhibiting the biofilm formation of P. aeruginosa (0.234 μl/ml) as evident from the Congo red dye reduction assay. Further the in vivo antibacterial efficacy of the black pepper oil nanoemulsion was tested on the shrimp (Penaeus monodon) and the MIC value of (0.234 μl/ml) was found effective in controlling the bacterial infection. Biosafety studies of blackpepper oil nanoemulsion on Artemia salina reveals the nontoxic nature of formulated nanoemulsion. The above findings envisage that black pepper oil nanoemulsion can be used as a potent antimicrobial agent for the control of bacterial infections in aquaculture industry. © 2018 Elsevier B.V.