Microbial infections are a general public health problem worldwide, and their resistance to antibiotics has increased rapidly. The prevention of microbial growth on the surface of cotton fabric is essential to control skin-related infections. In this work, we evaluated A.calamus- zinc oxide nanoparticle (AC-ZnONPs) coated cotton fabrics for antibacterial and cytotoxic activities. The formulated AC-ZnONPs were characterized by UV-spectrophotometer, SEM, EDX, FT-IR, and TEM studies. The AC-ZnONPs amalgamation into the cotton fabrics was done by the dipping technique. The antimicrobial activity and cytotoxic activity were executed by standard approaches. The results of UV-spectrophotometer, TEM, and SEM, and EDX proved the formation and existence of AC-ZnONPs on the fabrics' surface. FT-IR results demonstrated the existence of numerous functional groups. AC-ZnONPs efficiently suppressed the growth of the pathogenic microbes with a maximum inhibition at 60 μg of AC-ZnONPs against E.coli and S.aureus. The AC-ZnONPs coated fabrics also prevented microbial growth with maximum inhibition against P.aeruginosa, K.oxytoca, and A.baumanii. In addition, AC-ZnONPs efficiently suppressed the viability of SK-MEL-3 cells. Altogether, our findings disclosed that the AC-ZnONPs coated fabrics displayed excellent antimicrobial activity against the pathogenic microbes and could be utilized in the medical fields in the future.