In this paper the influence of different types of pretreatment (hydrogen peroxide and enzymes) on antimicrobial properties of knitted fabrics dyed with plant extracts of Achillea millefolium L. was examined. Three different cotton fabrics with different constructional characteristics and surface masses were used. Knitted fabrics were treated with methanolic plant extract (concentration 25 mg/mL) by using bath exhaustion method and subsequently treated with alginate. Antimicrobial properties of knitted fabrics treated with plant extract of Achillea millefolium L. on Escherichia coli, Staphylococcus aureus by using parallel streaking method (AATCC TM 147) were tested. In addition to antimicrobial properties testing, the mass loss of pretreated knitted fabrics, water retention value and color difference of pretreated and dyed knitted fabrics were also tested, depending on the type of applied pretreatment of knitted fabrics. It was found that the enzymatically treated knitted fabrics dyed with methanolic extract of the plant Achillea millefolium L. (concentration 25 mg/mL) and subsequently treated with alginate had better antimicrobial activity against Escherichia coli, compared to knitted fabrics treated with H2O2.