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In this study, the biosynthesis of polyhydroxyalkanoates (PHAs) was carried out using <i>Pseudomonas</i> putida and <i>Pseudomonas aeruginosa</i>. These PHAs were produced using reagent-grade glycerol and crude glycerol as the carbon sources. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production was obtained using <i>Pseudomonas putida</i> compared to <i>Pseudomonas aeruginosa</i>, and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the <i>Pseudomonas aeruginosa</i> strain.