As the global plastic consumption is increasing, an innovative substitute for petroleum-based product must be developed towards a more sustainable society. Cellulose is the most abundant biopolymer on earth having potential because of its biodegradability and is produced from renewable resources. However, purification of plant cellulose is costly and limit the application development where bacterial cellulose have gained more focus. Bacterial cellulose can be obtained as a by-product from Kombucha fermentation on commercial sugars such as sucrose. Replacing commercial sugars with molasses, a by-product from the sugarcane industry, as a substrate could be a low-cost alternative. The purpose of the study is to investigate and evaluate the properties of bacterial cellulose obtained from Kombucha microorganisms fermented in molasses medium using different fermenting conditions. Biofilms containing bacterial cellulose were obtained by fermenting with three types of molasses inoculated with two types of Kombucha cultures. Two of the three molasses used came from PT. Andalan Furnindo in Indonesia from two different sugar production batches and one type from Nordic Sugar AB in Sweden. The culture was obtained from previously fermented Kombucha, one from Tujju Kombucha, Indonesia and one from Roots AB, Sweden. Fermentation was carried out with one of the molasses from PT. Andalan Furnindo together with coffee waste, containing different concentrations of the substrates. The obtained biofilms were compared to biofilms produced with the ingredients of a conventional Kombucha setup. The other molasses from PT. Andalan Furnindo was fermented with or without the addition of pure caffeine, using culture with adapted microorganisms. Obtained films were either dried under pressure, without pressure in oven or purified with 1 M NaOH and air-dried. Optimum fermentation conditions with the molasses from Nordic Sugar AB were analysed. Regarding the fermentation with molasses from PT. Andalan Furnindo and coffee waste, the highest yield of biomass after fermentation could be seen in the system containing the highest amount of total sugars (100 g/l) and highest amount of caffeine deriving from coffee waste (200 mg/l). However, the membrane produced from conventional Kombucha exhibited a more flexible character, having superior elongation at break, stretching 46 % more than the sample produced in molasses medium. Using culture with adapted microorganisms in the fermentation with molasses from PT. Andalan Furnindo proved to increase the biomass yield with roughly 40 % compared to the biofilms produced without adapted microorganisms, but no effect of higher caffeine concentration was detected for the setups. All biofilms obtained from fermenting with adapted culture possessed superior mechanical-thermal properties. The highest elongation at break of 48.7 % was observed for the sample dried under normal conditions and the highest tensile strength was observed for the purified samples of 43.5 MPa. Furthermore, the purified samples possessed a higher thermal stability and had the highest cellulose content of 64 %. Adaptation was vital to obtain any bacterial cellulose fermenting in medium containing molasses from Nordic Sugar AB.