Bacteriophages and phage-derived lytic enzymes are emerging as promising alternatives to antibiotics in aquaculture; however, their effects on non-target microbial communities remain insufficiently characterized. This study evaluated the impact of a lytic phage (CH20) and a phage-derived lysin (LysVp1) on the microbiota associated with seawater, rotifers, and zebrafish larvae challenged with Vibrio alginolyticus GV09. Parallel treatments with phage CH20, lysin LysVp1, or no treatment were independently applied to each biological matrix. Microbial commu-nities were analyzed using 16S rRNA gene sequencing, with DNA- and RNA-derived datasets evaluated separately. Alpha diversity indices were compared using appropriate statistical tests, while beta diversity was assessed using Aitchison distance, PERMANOVA, and dispersion anal-yses, and differential abundance was evaluated using ANCOM-BC2. Alpha diversity metrics (Chao1, Shannon, and Simpson indices) showed no significant differences among treatments across all matrices, indicating preservation of microbial richness and diversity. Beta diversity pat-terns differed according to the nucleic acid source, with RNA-based analyses revealing treat-ment-associated shifts in rotifer and water microbiota that were not consistently detected at the DNA level. In zebrafish larvae, neither phage nor lysin treatment significantly altered overall community structure, although dispersion effects reflected limitations related to sample size. Overall, these results indicate that phage CH20 and lysin LysVp1 exert minimal impact on alpha diversity and limited, context-dependent effects on microbial community structure, supporting their microbiota-safe potential for aquaculture applications.