In the present study, the nano-silver-doped flax fibers (NAgDFF) are prepared in two steps. In the first step, oxidation of the flax fibers is performed by potassium periodate to form dialdehyde cellulose (DAC) and the second step is the reduction of silver ions by DAC. A series of characterization techniques of the photocatalyst NAgDFF was carried out using scanning electron microscopy, Fourier transform infrared spectroscopy, N2 adsorption isotherm, thermogravimetric analysis and energy-dispersive X-ray spectroscopy. The dye degradation potential of NAgDFF for methylene blue (MB), crystal violet (CV) and brilliant green (BG) (individually or mixture) was investigated using batch and column tests. The degradation efficiency was studied under optimized conditions such as pH (5.0), dye initial concentrations (100 ppm for MB and BG, and 150 ppm for CV), contact time (3.0 h), photocatalyst NAgDFF dose (0.08 g) and temperature (25° C). The maximum degradation efficiency of NAgDFF for MB, CV and BG is 64.75, 94.98 and 63.87 (mg/g), respectively. The kinetic studies show that the experimental data match well with the pseudo-second-order kinetic model. Furthermore, equilibrium isotherm data were analyzed according to Langmuir, Freundlich and Dubinin–Radushkevich equations. The thermodynamic parameters for the adsorption processes of cationic dyes on the NAgDFF fibers were also calculated; the negative value of ΔG° indicated the spontaneous nature of sorption. NAgDFF fibers were successfully applied for photodegradation of the investigated cationic dyes from different samples. The study was extended to investigate the biological activity of newly synthesized NAgDFF against various microorganisms.