Biosynthesis of silver nanoparticles (AgNPs) using bark extract of Acacia nilotica as green reducing agent and natural colorant was achieved. Simultaneous dyeing of Egyptian cotton fabric and control of the in vitro contamination of Egyptian cotton culture (var Giza 86) plant were also carried out. Ferric chloride was used as metal mordant in the dyeing process and the color measurements were carried out to evaluate the shade obtained. The dyed fabric was subjected for analysis in terms of K ̸S, CIE L*a*b*, and fastness properties. For assaying the antimicrobial activity, the disk diffusion assay on two model organisms, E. coli (Gram Negative) and Bacillus subtilis (Gram-positive) were achieved resulting that the treated cotton fabric showed a clear antimicrobial against both model organisms. The treatments of produced AgNPs on cotton not only improve its antimicrobial efficiency but also influenced the tensile strength of the fabric sample positively. The treatment was found to enhance the color depth and fastness properties of dyed cotton fabric samples. Concerning the new application for in vitro control of bacterial and fungal contamination, the synthesized AgNPs was conducted in the sterilization protocol and showed the effective elimination of fungal and bacterial contamination in in vitro cotton var G86, these results provided a new sterilization product to be used for future studies of plant tissue culture.