This study evaluated single-cell protein production from PHA-rich mixed microbial cultures obtained from fermentation and subsequent PHA storage, using urban (namely food waste and municipal sewage sludge; FW-MSS) and agricultural waste (namely wine lees; WL) streams as substrates. FW-MSS fermentation achieved stable short-chain fatty acid (SCFA) production and a high CODSCFA/CODSOL ratio of 0.77 ± 0.01, which allowed to select a mixed microbial culture (MMC) with intracellular PHA content of 15.1 wt%, which aligns with fish dietary standards and yielded a MMC biomass with a protein level of 55.1 wt% and a balanced essential amino acid (EAA) profile. In contrast, WL fermentation showed lower SCFA content and stability, yielding a MMC with 45.8 wt% of protein along with a high non-conformance rate (53.65 %), and 7.2 wt% PHA, making the resulting MMC more suited as a supplemental protein source. Distinct microbial communities developed in the two SBRs due to different feedstocks, influencing the abundance of PHA-storing bacteria, with no known fish pathogens detected in either sample. Statistical analysis confirmed FW-MSS’s superior product consistency, supporting its potential as a good quality SCP for aquafeed, especially for rainbow trout, as confirmed by its high essential amino acid index (EAAI).