•BC was produced by Komagataeibacter rhaeticus bacterium isolated from Kombucha tea.•Sugarcane molasses (SCM) was used as alternative culture medium.•The increase of SCM in culture media led to smooth and flexible BC membranes.•The alternative culture medium favors reducing costs and enhancing BC production. Komagataeibacter rhaeticus, a bacterium isolated from Kombucha tea, was used to produce bacterial cellulose (BC) through its cultivation in a static sugarcane molasses (SCM) supplemented-culture medium (totally or partially), as an alternative carbon source. BC membranes were characterized by different physicochemical analysis using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission gun-scanning electron microscopy (FEG-SEM), thermogravimetry analysis (TGA) and PeakForce quantitative nanomechanics atomic force microscopy (PeakForce (QNM-AFM)). FTIR, XRD and TGA results suggest great similarity among all membranes produced by distinct culture media. Although the glucose (F1) and SCM (F6) media presented the lowest BC yield, all SCM-supplemented culture media (from F2 to F5) showed BC yield values similar to the HS culture medium (F0). FEG-SEM analysis showed that as higher SCM concentrations on culture media higher dense nanofibers network could be prepared. Quantitative nanomechanical results obtained by AFM technique corroborate FEG-SEM analysis besides show smoother and more flexible BC membranes as a function of the increasing of the SCM concentrations. The modification of the carbon source of the culture medium with an important by-product of Brazilian agroindustry appears as a viable alternative to reduce cost of BC production (of up to 20.06%) besides increase the possibilities of industrial scale BC preparation.