Sodium cellulose sulfates were prepared in various degrees of substitution (DS) and degree of polymerization (DP) by directly sulfating cotton and microcrystalline cellulose. Molecular parameters of the prepared samples were characterized using elemental analysis, IR spectroscopy, and ¹³C NMR spectroscopy techniques. The antibacterial activities of the sodium cellulose sulfates were assayed against both gram-negative and gram-positive bacteria. The study examined the dependence of antimicrobial properties on molecular weight (Mw) and DS. The samples showed DS- and DP-dependent antibacterial activity against both gram-negative and gram-positive pathogenic microorganisms. Samples with DS ≥2.0 exhibited the highest antibacterial activity, indicating the presence of sulfate groups in C-2 and/or C-3 carbon atoms in cellulose anhydroglucose unit (AGU) induces the antibacterial activity. Additionally, SCS samples with DP around 340 showed the highest antibacterial activity against all species of bacteria. The mechanism of antimicrobial effect of SCS derivatives is most likely directed at the destruction of bacterial membranes.