The halophilic bacteria Bacillus cereus LB7 and Burkholderia gladioli 2S4R1 were found utilizing mixed sugars which are released upon saccharification of paddy straw. The C:N ratio for growth and PHB production by LB7 and 2S4R1 was optimized at 30:1 and 38:1, respectively and a novel growth media (PMS-NaCl) containing saccharified paddy straw leachate as the sole C source was formulated. The PMS-NaCl was used for studying the growth kinetics and PHB production rate. At optimized parameters, B. cereus LB7 and B. gladioli 2S4R produced PHB @ 0.387 g L−1 h−1 and 0.309 g L−1 h−1, respectively which was statistically at par with the PHB yield obtained by utilizing pure glucose as the sole C source. Based on findings, it is suggested that paddy straw hydrolysates can be used as a substrate to produce PHB by halophilic bacteria and explored further for industrial-scale applications. Bacterial production of Poly-β-hydroxybutyrate using saccharified paddy straw leachate as a substrate. Display omitted •Utilization of paddy straw as potential feedstock for PHB production.•PHB production by halophilic bacteria by using saccharified paddy straw leachate.•Effect of C:N ratio and bacterial growth stage on PHB production.•Alkali pretreatment and enzymatic saccharification of paddy straw.