Red mineral pigment use is recognized as a fundamental component of a series of traits associated with human evolutionary development, social interaction, and behavioral complexity. Iron-enriched mineral deposits have been collected and prepared as pigment for use in rock art, personal adornment, and mortuary practices for millennia, yet little is known about early developments in mineral processing techniques in North America. Microanalysis of rock art pigments from the North American Pacific Northwest reveals a sophisticated use of iron oxide produced by the biomineralizing bacterium Leptothrix ochracea; a keystone species of chemolithotroph recognized in recent advances in the development of thermostable, colorfast biomaterial pigments. Here we show evidence for human engagement with this bacterium, including nanostructural and magnetic properties evident of thermal enhancement, indicating that controlled use of pyrotechnology was a key feature of how biogenic iron oxides were prepared into paint. Our results demonstrate that hunter-gatherers in this area of study prepared pigments by harvesting aquatic microbial iron mats dominated by iron-oxidizing bacteria, which were subsequently heated in large open hearths at a controlled range of 750 °C to 850 °C. This technical gesture was performed to enhance color properties, and increase colorfastness and resistance to degradation. This skilled production of highly thermostable and long-lasting rock art paint represents a specialized technological innovation. Our results contribute to a growing body of knowledge on historical-ecological resource use practices in the Pacific Northwest during the Late Holocene.Figshare link to figures: https://figshare.com/s/9392a0081632c20e9484.