Milkfish, an important aquaculture species in Asian countries, are traditionally cultured in outdoor-based systems. There, they experience potentially stressful fluctuations in environmental conditions, such as temperature, eliciting changes in their physiology and metabolism. While the importance of the gut microbiome for the welfare and performance of fish has been studied previously, little is known about the effects of thermal stress on the gut microbiome of milkfish and its interactions with the host’s metabolism. In this study, we investigated the gut microbiome of juvenile milkfish in a thermal stress experiment (control treatment: 26°C, high temperature: 33°C). Fish were sampled for 16S amplicon sequencing (V4-V5 hypervariable region) of the gut content at the beginning of the experiment (d0 after the gradual increase to 33°C in the high temperature treatment), after 14 days (d14), and at the end of the experiment after 21 days (d21). Additionally, physiological data on stress response and metabolic biomarkers were collected. To assess the influence of the fish feed or the tank water on the milkfish gut microbiome, feed pellets and water filters (0.2µm pore size) were likewise sequenced. However, the feed samples did not contain any amplifiable DNA and are therefore not included in this submission. Scripts for the bioinformatic and statistical analysis as well as the final OTU table are archived alongside the physiological data on Pangaea. This study is part of the ACUTE project (AquaCUlture practice in Tropical coastal Ecosystems - Understanding ecological and socio-economic consequences) funded by the Leibniz Association grant SAW-2015-ZMT-4.