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An antibacterial agent (MgO2) was synthesised using 0.2 and 0.4 M concentrations of MgCl2·6H2O and H2O2, which was subsequently applied to cotton fabric using a conventional pad-dry-cure method in order to achieve antibacterial properties against S. aureus and E. Coli microorganisms. The antibacterial effect against these microorganisms was investigated using a zone of inhibition test and the percent reduction method. The outcomes of these measurements showed that when the cotton fabric was treated with the reaction product of MgCl2·6H2O and H2O2, it retained 90–93% antibacterial activity against S. aureus and 89–91% against E. coli bacteria. This antibacterial effect against these microorganisms was attributed to the presence of reactive oxygen species and Mg ions on the treated cotton fabric. Long term antibacterial effects against S. aureus and E. coli microorganisms were recorded for up to 70 laundering cycles, and the amounts of retained bound peroxide and Mg ions on the finished specimens were measured using iodimetric titration and MP-AES measurements. Additionally, the properties of synthesised MgO2 crystalline powder and treated cotton fabric were studied using UV–Vis, EDX, FTIR spectroscopy, and SEM measurements. The influence of the MgO2 application on mechanical properties such as tensile strength, tear strength, whiteness index, and crease recovery angle of the treated cotton fabric was also analysed. The results obtained clearly confirmed that the treated cotton fabric possessed antibacterial effects for up to 70 laundering cycles. This is likely due to the presence of the required amount of oxidative species and Mg ions on the treated cotton fabrics. The FTIR and EDX results showed that the presence of these key elements (oxygen containing groups) was responsible for the antibacterial property of the finished fabrics. The whiteness index and tensile strength were improved after treatment with MgO2, although tear strength and flexibility of treated specimens were decreased after treatment.