In the present study, bacterial nanocellulose/graphene oxide nano-biocomposites (BNC-GO-NBCs) were fabricated by Komagataeibacter saccharivorans NUWB1 using an in-situ method involving three time-dependent approaches. Physicochemical studies showed that the chosen dried BNC-GO-NBC possessed a three-dimensional interconnected porous structure of BNC with GO layers embedded within the BNC fibrils. BNC-GO-NBC had a crystallinity index of 74.21 %, higher thermostability up to 380 °C and could withstand a tensile load of 84.72 MPa. N2 adsorption-desorption isotherm of the BNC-GO-NBC was found to be of type IV, suggesting a mesoporous type structure with a total pore volume and surface area of 6.232e-04 cc g−1 and 10.498 m2. BNC-GO-NBC exhibited remarkable adsorption capacity for two cationic dyes, Rhodamine B (RhB) and Acridine Orange (AO), and the adsorption data conformed well to the Langmuir isotherm (R2 = 0.99) and pseudo-second-order model. Thermodynamic studies indicated that the adsorption process was spontaneous and endothermic. Additionally, the BNC-GO-NBC displayed the potential for regeneration, with the ability to be recycled up to five times. Further, the antibacterial activity, cell cytotoxicity and oxidative stress assays of the BNC-GO-NBC revealed its non-cytotoxic nature. The findings of the present investigation evidently suggest the potentiality of BNC-GO-NBC in the application of dye adsorption and other environmental applications.