The development of new materials to be used as a substrate for plant tissue culture can lead to substantial advances in biotechnology. Here, mats consisting of a mixture of nonwoven (randomly oriented) and aligned nanofibres were produced by electrospinning solutions of polylactic acid (PLA) and polyvinylidene fluoride (PVDF). They were referred to as PLA4 and PVDF4, respectively. Callus initiation from stem explants of bilimbi (Averrhoa bilimbi L.) and aromatic chilli (Capsicum frutescens L.) occurred on these two types of nanofibre mats floated in liquid Murashige and Skoog (1962) basal medium supplemented with 2 mg/l α-naphthalene acetic acid or 2,4-dichlorophenoxy acetic acid, respectively. After subculturing for 3 weeks, the fresh weight of callus initiated from stem explants of bilimbi was significantly greater by 20% when PVDF4 rather than PLA4 nanofibre mats were used as a support matrix. In contrast, both the fresh and dry weights of callus initiated from stem explants of aromatic chilli were significantly greater by 11% and 50%, respectively, when PLA4 and not PVDF4 nanofibre mats was used. These differences in response to the two types of nanofibre mats were also found in relation to growth of callus of bilimbi and aromatic chilli during subculture. This is the first time electrospun nanofibre mats have been used in plant tissue culture research.