Polyhydroxyalkanoates (PHAs), environmental friendly polyesters, can be produced by a wide range of microorganisms in nutrient‐limiting conditions. In the present study a new bacterial strain, able to produce high quantities of poly‐3‐hydroxybutyrate (PHB), is isolated from contaminated soil. The isolate is characterized and identified as Bacillus cereus Y23, based on morphological, physiological characteristics, and 16S ribosomal RNA gene sequences. PHB production is evaluated using different carbon and nitrogen sources and nutritional and culture conditions are optimized. The highest PHB production of 5.12 g L−1 was obtained in growth medium containing fructose and ammonium sulfate at a C/N ratio of 30, 0.3% yeast extract, 4.5 g L−1 phosphate solution, pH 7, inoculum size of 0.3%, and agitation rate of 150 rpm after 48 h of incubation at 32 °C. The PHB production decrement can be attributed to the intracellular utilization of the polymer during the stationary phase of the cellular growth and to the sporulation process. The presence of the main functional groups of PHB polymer is verified by Fourier‐transform infrared analysis. At optimum conditions, B. cereus Y23 turned out to be a good candidate for industrial production of PHB since it accumulates up to 67.9% of its dry weight.