Polyhydroxyalkanoates (PHAs) are bioplastics derived from renewable resources such as vegetable oils, corn starch, or microbes. The polyhydroxybutyrate (PHB) is a short-chain-length PHA, and the most important bioplastic produced by certain microorganisms in the presence of excess carbon sources. In this study batch cultivation of Alcaligenes eutrophus with the aim of increasing PHB production using different carbon and nitrogen sources was performed. The accumulation of PHB granules in this strain was significantly dependent on the type of carbon and nitrogen sources in the culture medium. The bacteria were cultivated on various carbon sources including glucose, fructose, lactose, lactic acid, arabinose and sucrose and nitrogen sources including ammonium chloride, ammonium sulfate, peptone, urea and tryptone at constant concentrations, temperature and pH. Cell growth and PHB production were quantified by measuring absorbance at 600 nm and 235 nm (absorbance of crotonic acid), respectively. The best results were obtained when using fructose and ammonium chloride as carbon and nitrogen sources, with a carbon/nitrogen ratio of 10. The production of PHB was growth associated as indicated by the growth and PHB production kinetics. Atomic force microscopy analysis of PHB film also showed high porosity of PHB recovered by chloroform.