The rising prevalence of antibiotic-resistant bacteria and biofilm formation in seafood presents significant public health risks. This study examined 90 fish samples, including brush tooth lizard (Saurida undosquamis), red porgy (Pagrus pagrus), and mullet (Mugil cephalus) (30 of each), to investigate the anti-biofilm properties of essential oils derived from cumin and lemongrass against V. parahaemolyticus at varying temperatures (4 °C and 37 °C). Additionally, the study assessed virulence-encoded genes in V. parahaemolyticus and the susceptibility of Vibrio isolates to antimicrobial agents. Bacteriological analysis identified 46 (51.1%) Vibrio species. Among these isolates, 36.8% carried the virulence-associated genes tdh, trh, and tlh. Antibiotic sensitivity testing revealed significant resistance among the isolates, with an average multiple antibiotic resistance (MAR) index of 0.473. V. parahaemolyticus showed markedly increased biofilm formation at 37 °C compared to 4 °C, highlighting a substantial temperature-dependent difference in production. Furthermore, lemongrass essential oil (LEO) and cumin essential oil (CEO) effectively inhibited V. parahaemolyticus biofilm production at sub-minimum inhibitory concentrations. These findings underscore the importance of regularly monitoring seafood for Vibrio species due to their antibiotic resistance, unique molecular traits, and biofilm-forming ability, all of which are critical for improving seafood safety. The demonstrated efficacy of LEO and CEO in inhibiting biofilm formation also presents a promising strategy for the food industry to enhance the safety and shelf-life of seafood products, potentially reducing the reliance on conventional antibiotics and addressing the growing challenge of antibiotic resistance. Overall, the study demonstrated that LEO and CEO could feasibly inhibit V. parahaemolyticus biofilm production and enhance the safety of consumed fish.