This study aimed to investigate the production of isomalto-oligosaccharides from banana flour in a batch reactor and to study the fermentation properties of the synthesised IMO by conventional yoghurt strains and selected probiotic strains. IMOs were synthesised from hydrolysed banana (Musa spp.) flour by the action of transglucosidase L enzyme (E.C. 3.2.1.20). Banana flour was liquefied by Termamyl SC (fungal α-amylase, E.C. 3.2.1.1), at 95oC, pH = 5.5-6.0, followed by saccharification by either Fungamyl 800L (fungal α-amylase, E.C. 3.2.1.1), at 50oC, pH = 5.5-6.0, or barley β-amylase (E.C. 3.2.1.2), at 45oC, pH 4.5-5.0. Both saccharified banana slurries were then used as substrates for IMO synthesis by application of transglucosidase L and comparable yields were obtained (approximately 30% total sugar). After 12 hours of reaction time the maximum IMO yields from Fungamyl 800L and barley β-amylase saccharified banana slurries were obtained (33.29% of total sugar or 7.67 ± 0.27% w/v, and 30.13% of total sugar or 7.06 ± 0.49% w/v, respectively). Glucose and maltose were removed by fermentation by 1% (w/v) bakers’ yeast after 24h, the final IMO mixture composed of 53% isomaltotriose/panose, 21% isomaltotetraose, and 26% maltooligoheptaose and larger oligomers. The synthesized IMO mixture was added to set yoghurt using two starter combinations (S. thermophilus and L. delbrueckii spp. bulgaricus, and L. acidophilus and L. casei). After 28 days of storage, the bacterial population changes were insignificant. Lactose was a more preferable carbon source than IMO in both yoghurt samples as approximately 50%and 40% (respectively) of IMO were depleted.