Due to the sudden pandemic outbreak, there is a substantial demand for antimicrobial textiles for health and hygiene. Natural dyeing with plant sources has been proven to be an excellent eco-friendly method for producing healthcare textiles. In this study, Diospyros mollis Griff. extract was applied to the simultaneous dyeing and multi-functionalization (antibacterial activity and UV-protection) of wool fabrics. Response surface methodology was applied to optimize the treatment procedure and assess parameter interactions. The optimal result was achieved when dyeing at pH 4, dye concentration 25 g/L, temperature 88°C, and time 95 min. The dyed fabrics had good antibacterial activity against both E.coli and S.aureus (bacterial colony reduction > 90%), with E.coli being more pronounced. The UV protection factor (UPF) also reached the maximum (40+) level, demonstrating their superior UV protection. The finished wools are black, with high color strength (K/S > 9), good light and washing fastness, and fair to good crocking fastness. Thus, Diospyros mollis Griff. fruit extract can be used as a new bioactive agent for multifunctional textiles, as well as simultaneous black coloring.