Growing concern over plastic pollution has intensified research on biodegradable alternatives, such as polyhydroxybutyrate (PHB), a biopolymer produced by cyanobacteria. Despite their sustainability advantages, photoautotrophic PHB production remains limited, and cultivation strategies need optimization. In this study, five cyanobacterial strains were isolated from environmental microbiome cultures to evaluate their PHB production potential. The goal was to identify the most productive strains and optimal conditions for polymer synthesis. Cultures were grown in modified BG11 media (without nitrogen, phosphorus, or inorganic carbon) and in a secondary effluent from treated urban wastewater, both supplemented with acetate (0, 0.6, or 4 g/L) and incubated for 7 days in darkness. The biomass remained stable in most strains but declined to 0.28 g/L in the secondary effluent, except for one Leptolyngbya sp. strain that increased the biomass with acetate. The highest PHB yield per acetate consumed was achieved by Synechocystis sp. from an agricultural pond, reaching 3.1% dry cell weight in modified BG11 with 0.6 g/L acetate. In the secondary effluent, the maximum PHB content reached 2.9% in another Leptolyngbya sp. strain with 4 g/L acetate. These findings highlight strain-specific responses and the potential of wastewater-based cultivation for sustainable bioplastic production.