Petroleum-based plastics have become a big problem in many countries because of their non-degradability and that they become microplastics in the environment. This study focused on the optimization of production medium and conditions of polyhydroxyalkanoates (PHAs), which are biodegradable bioplastics and are accumulated in microbial cells. Among 341 isolates from 40 composted soil samples, the best isolate was the HD34 strain, which was identified using morphological, molecular, and biochemical methods. The results showed that the strain was most closely related to Ensifer adhaerens LMG20216T, with 99.6% similarity. For optimization of production medium and conditions using response surface methodology, it exhibited an optimal medium containing 3.99% (w/v) of potato dextrose broth (PDB) and 1.54% (w/v) of D-glucose with an adjusted initial pH of 9.0. The optimum production was achieved under culture conditions of a temperature of 28 °C, inoculum size of 2.5% (v/v), and a shaking speed of 130 rpm for 5 days. The results showed the highest PHA content, total cell dry weight, and PHA yield as 72.96% (w/w) of cell dry weight, 9.30 g/L, and 6.78 g/L, respectively. The extracted PHA characterization was studied using gas chromatography, 1H NMR, FTIR, and XRD. The results found that the polymer was a polyhydroxybutyrate (PHB) with a melting temperature (Tm) and degradation temperature (Td) of 173.5 °C and 260.8 °C, respectively.