The accumulation of plastic waste in the environment has become a serious environmental problem worldwide. Biodegradable plastics, such as polyhydroxyalkanoate (PHA), could serve as green alternatives to petroleum-based plastics. In this study, a mixed microbial culture was enriched under feast/famine conditions using a sequencing batch reactor (SBR) with acetate as a carbon source. The enrichment could accumulate a maximum PHA concentration of 32.3% gPHA/g mixed liquor suspended solids (MLSS) in the 12th cycle of SBR operation. The microbial community in this sludge sample was analyzed using 16 S rRNA gene amplicon sequencing (MiSeq). The results showed the dominance of Proteobacteria, represented by Alphaproteobacteria (13.26% of total sequences), Betaproteobacteria (51.37% of total sequences), and Gammaproteobacteria (23.44% of total sequences). Thauera (Betaproteobacteria) had the highest relative abundance, accounting for 48.88% of the total sequences. PHA-accumulating microorganisms in the enrichment were detected using fluorescence in situ hybridization (FISH) and a fluorescent dye, Nile blue A. Alphaproteobacteria and Betaproteobacteria were capable of accumulating PHA, while no Gammaproteobacteria were detected. Thauera spp. from Betaproteobacteria constituted 80.3% of the total PHA accumulating cells.