Temperature is a critical environmental factor influencing fish physiology and health, yet its effects on host-associated microbial communities remain poorly understood. In fish, the skin microbiome plays a critical role as a barrier and in immune defense, making it particularly vulnerable to thermal stress. In this study, we investigated the impact of acute heat stress on the skin-associated microbial community of rainbow trout (Oncorhynchus mykiss). Fish were exposed to short-term heat stress (from 15 °C to 24 °C), and skin samples were collected before and after heat exposure. Along with other physiological and immunological parameters, the microbial community was analyzed using high throughput nanopore DNA sequencing. Acute heat stress resulted in a significant shift in microbial community structure, with an increase in the relative abundance of opportunistic or stress-tolerant taxa, including Flavobacteriaceae and Enterobacteriaceae families. These findings demonstrate that even short-term thermal stress can rapidly disrupt the skin microbiome of rainbow trout, potentially compromising host health. Understanding how environmental stressors influence host–microbe interactions is increasingly important in the context of climate change and aquaculture sustainability.