The objective of this study was to evaluate the effect of modified atmosphere packaging (MAP) on fish skin, gills and intestines bacterial microbiome.Whole gilthead seabream was packed aerobically or under modified atmospheres (60% CO2, 30% N2, 10% O2) and stored isothermally at 0 °C. Next Generation Sequencing (NGS) analysis was applied for the characterization of fish microbiome on fish skin, gills, and intestines initially (time of packaging, 1 day after harvesting) and after 10 days of isothermal storage at 0 °C. NGS results indicated statistically significant differences in families’ richness and diversity in the tested fish tissues between aerobic and MAP packaging during storage at 0 °C. The most persistent bacteria were Proteobacteria for both packaging types. For fish skin microbiota, the initially prevailing families were Comamonadaceae, Enterobacteriaceae and Moraxellaceae while in the intestines Comamonadaceae, Anaplasmataceae, Bacillaceae and Enterobacteriaceae were the dominant bacteria. At the end of storage period, the fish microbiota was dominated by psychotropic and psychrophilic families (Pseudoalteromonadaceae, Psychromonadaceae, and Shewanellaceae), while families such as Comamonadaceae were persistent under MAP conditions. By 8 days of fish storage at 0 °C, ΜΑΡ samples exhibited higher sensory scorings than the respective aerobically stored fish, indicating better retention of fish freshness and fish quality attributes under MAP. Based on the results of the study, MAP modified significantly the microbiological status and extended the shelf life of fish. NGS was a powerful tool that provided a more complete assessment compared to a culture-based analysis.