Abstract Poly(3-hydroxybutyrate), PHB, and poly(3-hydroxybutyrate- co -4-hydroxybutyrate), P(3HB -co- 4HB), consisting of 0–94% mole fraction of 4HB content, were produced in high content by Cupriavidus necator strain A-04. The carbon sources used for PHB production included sugars made locally in Thailand: refined sugarcane, brown sugarcane, rock sugar, toddy palm sugar and coconut palm sugar. The switching of the ratios of carbon to nitrogen, together with the ratios of fructose to 1,4-butanediol, were applied to P(3HB- co -4HB) production in fed-batch cultures. Optimal P(3HB- co -4HB) production was achieved with 112 g biomass and 73 g P(3HB- co -4HB) with 38% mole fraction of 4HB content. Next, P(3HB -co- 4HB) with a 0, 5, 24, 38 and 64% mole fraction of 4HB content were purified and prepared as plastic films. The mechanical properties and biocompatibility of these films were tested and compared with commercial PHB, polystyrene (PS) and polyvinylchloride (PVC) prepared without additives. The results demonstrated that PHB had thermal and mechanical properties similar to those of commercial PHB. The P(3HB- co -4HB) polymers possessed melting temperature and glass transition temperature values higher than those reported previously. The mechanical properties were compared with those of PS and PVC. The in vitro biocompatibility was assessed using L929, human dermal fibroblast and Saos-2 human osteosarcoma cells. The cytotoxicity results and scanning electron micrographs showed that P(3HB -co- 4HB) films have good surface characteristics and can promote cell attachment, proliferation and differentiation. Combined with their good mechanical properties, P(3HB- co -4HB) polymers possess potential usefulness for biomaterial applications in artificial skin tissue support and orthopedic support.