This paper focuses on assessing some characteristics (including permeability of fabrics to air, moisture water vapor absorption, weight per unit area, thermal stability, morphology) and antibacterial ability of the cotton fabric dyed with the extract from fresh Diospyros mollis (DM) Griff fruit. The aqueous DM fruit extract was applied to modified cotton fabrics. Tannins, hydroquinone, and saponins—three main components in the aqueous DM fruit extract were determined using weight method and HPLC method. The ratio of fresh DM fruits/distilled water of 1/2 (m/v) was against well to Escherichia coli and Staphylococcus aureus strains with the killed bacteria percentage of 96.65 and 92.29%, respectively. The change in properties of cotton fabrics after dyeing was assessed and compared to that of the undyed sample. Especially, the antibacterial ability to Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) strains of dyed cotton fabrics after washing cycles was tested. The obtained results showed that there were some positive changes in the air permeability, moisture water vapor absorption, and weight per unit area of the dyed cotton fabric while the thermal stability of dyed cotton fabric was not affected by dyeing process. The antibacterial ability of dyed cotton fabric was reduced when increasing the number of washing cycles. Moreover, the color fastness to rubbing, washing and light of dyed cotton fabrics reached high rating, rating of 3/5, 4/5 and 7/8, respectively. The effect of technological factors has been optimized using Minitab software with Design of experiments (DOE) by response surface design. The optimal conditions were dyeing time of 90 min, dyeing temperature of 56.56 °C and the ratio of DM extract/water of 89/100. At this condition, the dyed cotton fabrics could kill 99.9% E. coli and 99.9% S. aureus. In general, this dyed cotton fabric has potential applications in the garment, medical and other fields thanks to its excellent antibacterial ability.