The pervasive utilization of plastics and their integration into ecosystems has resulted in significant environmental issues, particularly the pollution of microplastics (MPs). In aquaculture, high-fat feed (HFD) is frequently employed to enhance the energy intake and economic fish production. This study utilized zebrafish as a model organism to investigate the impact of concurrent exposure to HFD and MPs on fish intestinal pathology damage and intestinal microbiome. The experimental design involved the division of zebrafish into two groups: one receiving a normal diet (ND) and the other receiving HFD. The zebrafish were exposed to a control group, as well as polystyrene (PS) MPs of varying sizes (5 and 50 μm). Histopathological examination revealed that the combination of 5 μm MPs and HFD resulted in the most significant damage to the zebrafish intestinal tract. Furthermore, gut microbiome assays indicated that exposure to MPs and HFD altered the composition of the gut microbiome. This study demonstrates that in aquaculture, the issue of HFD must be considered alongside concerns about MPs contamination, as both factors appear to have a combined effect on the intestinal pathology damage and intestinal microbiome. The findings of this research offer valuable insights for the improvement of fish farming practices.