In the present study, the PHAs production efficiency of a mixed culture rich in PHA-forming bacteria, and bacterial strains isolated from it, was examined under stress conditions. Nitrogen and dual nitrogen–oxygen limitation was applied, using both synthetic and OMW, as the sole carbon source. The scope of those experiments was to compare the ability of those cultures to produce PHAs and to investigate whether a PHA overproducing strain was isolated. Under stress conditions such as nitrogen limitation bacterial growth is restricted and carbon flux is therefore driven to PHA formation. On the other hand, oxygen limitation usually occurs during the fermentation process thus dual nitrogen–oxygen limitation was applied in order to investigate the additional effect of oxygen limitation in our study. In addition, we studied the valorization of OMW, a common waste stream in the Mediterranean region, via its microbial transformation to PHAs using the enriched culture and its isolates.