Substitution of aquaculture feed ingredients to reduce reliance on capture fisheries may affect gut microbiome and resilience to environmental stress. To investigate replacement protein sources in feeds for black tiger prawn, Penaeus monodon, three isonitrogenous, isoenergetic diets were tested in a randomised blind trial using fisheries by-product meal, cattle and poultry by-product meal and soybean meal as the major protein. Juvenile P. monodon (1.46 ± 0.28 g, 24 per tank, 4 tanks per treatment) were acclimatised on reference diet for 2 weeks, weighed, then fed experimental diets for two weeks. Elevated temperature, pH and salinity was applied for a further two weeks. Feed conversion ratio was significantly higher in animals fed soybean meal-based diet (F (2,9) = 21.7, p < 0.001). To determine any effect of diet on intestinal microbiome the V6-V8 region of the 16s rRNA gene was sequenced on arrival from the farm, at the end of acclimatisation, 24 h after starting test feeds, after 2 weeks on test feeds and after stress period. We found no significant effect of diet on alpha diversity of the midgut microbiota, and no correlation between stress resilience, diet and gut microbiome diversity. However, time had a significant impact on beta diversity throughout the experiment across all three diets (p < 0.001), with the most abundant genera on arrival from the farm being from the genera Photobacterium and Vibrio while the relative abundance of members of the Rhodobacteraceae, Pseudoalteromonadaceae, Mycoplasmataceae and Flavobacteriaceae increased throughout the trial and were also found in the seawater supply. Changing only the protein in the diet is not sufficient to impact gut microbiome, even when animal growth and feed conversion is severely compromised by the diet. We found that time/age is the major driver of intestinal microbiome changes in P. monodon, with diversity increasing as the animal ages.