Antimicrobial resistance is a significant concern in aquaculture, prompting the exploration of nanoemulsions (NEs) with a non-resistant mode of action as a safer and more effective alternative to antibiotics. In Asian countries, the culture of Penaeus vannamei has faced growing concerns as V. parahaemolyticus (Vp) has been increasingly linked to shrimp diseases, leading to huge economic losses. In response, stable ozonated oil-in-water NEs (NE-25, 26, 28, 29) were produced using a microfluidization method and characterized by their polydispersity index and average droplet size (NE-25, 26, 28, 29), which were found to 0.108, 0.251, 0.223, 0.173, and 229.7nm, 190.2nm, 201.8nm, and 145.8nm, respectively. The therapeutic potential of NEs is tested against pathogenic strains having tdh and toxR virulence gene, which were isolated from diseased shrimp (VP-S7 and VP-S14), these were found to be more resistant compared to commercial V. parahaemolyticus (MTCC-451) strain. Our NEs confirmatory antibacterial tests include bacteriostatic, bactericidal, antibiofilm, adherence, live/dead assays, and oxygen consumption rate (OCR) studies. All tested nanoemulsions showed effectiveness against planktonic and biofilm stages of V. parahaemolyticus; NE-25 showed strong adherence inhibition, with results of VP-S7 (49.55%), VP-S14 (53.47%), and MTCC-451 (68.76%). The biofilm inhibition of NE-25 (55.97%) is comparable to inhibition with antibiotics gentamicin (58.73%). The NE OCR results (1 × 107 cell/ml) compared to antibiotics treated and untreated, the fold reduction for NE-28 (0.2) is highly significant when compared with commercial antibiotics (0.6) and the growth control (1.0). Therefore, using NEs, which possess high antibacterial properties, is a promising alternative for treating V. parahaemolyticus compared to current antibiotics.