The present work established the optimization and production of biodegradable thermoplastic polyhydroxybutyrate (PHB) from Amazonian microalga Stigeoclonium sp. B23. The optimization was performed in eight different growth media conditions of Stigeoclonium sp. B23, supplemented with sodium acetate and sodium bicarbonate and total deprivation of sodium nitrate. B23 was stained with Nile Red, and PHB was extracted and quantified by correlating the amount of fluorescence and biopolymer concentration through spectrofluorimetry and spectrophotometry, respectively. Our results detected the production of PHB in Stigeoclonium sp. B23 and in all modified media. Treatment with increased acetate and bicarbonate and without nitrate gave the highest concentration of PHB, while the treatment with only acetate gave the lowest among supplemented media. Our results showed a great potential of Stigeoclonium sp. B23, the first Amazonian microalga reported on PHB production. The microalga was isolated from a poorly explored and investigated region and proved to be productive when compared to other cyanobacterial and bacterial species. Additionally, microalga biomass changes due to the nutritional conditions and, reversely, biopolymer is well-synthetized. This great potential could lead to the pursuit of new Amazonian microalgae species in the search for alternative polyesters.