Fluorescent protein-tagged bacterial strains are widely used tools for studying host-pathogen interactions and microbial dynamics. In this study, we developed and characterized Vibrio harveyi strains genetically modified to express green fluorescent protein (GFP) and red fluorescent protein (RFP). These strains were constructed using triparental mating and evaluated for phenotypic, genomic, and virulence attributes. Genomic analyses revealed strain-specific variations, including mutations in key regulatory and metabolic genes, such as luxO and transketolase. While plasmid acquisition imposed metabolic costs, resulting in altered growth and antibiotic sensitivities in certain transconjugants, others demonstrated robust phenotypic stability. Virulence assays using gilthead seabream larvae revealed that most tagged strains retained moderate pathogenicity, with visualization of co-infections highlighting the potential for studying strain-specific interactions. Furthermore, fluorescent microscopy confirmed the successful colonization and localization of tagged bacteria within host tissues. These findings underscore the utility of GFP- and RFP-tagged Vibrio harveyi as versatile tools for infection dynamics, offering a foundation for future research on strain interactions and pathogen-host relationships.