Aim: The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. Methods and Results: In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l(-1) sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) - P(HB-co-HV) - of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l(-1)) in a 3.0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2.7 g l(-1), respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2.5 g l(-1). The polymer was characterized by proton NMR. Conclusions: Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. Significance and Impact of the Study: Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production.