In this study, a stimuli-responsive cotton fabric with controlled antimicrobial properties was prepared using temperature- and pH-responsive microgels based on poly(N-isopropylacrylamide) and chitosan (PNCS microgel) in combination with biological-barrier (bio-barrier) forming polysiloxane matrices. For the polysiloxane matrix, (3-aminopropyl)triethoxysilane (APTES) was used in concentrations of 0.5, 2 and 4%. For comparison, 3-(trimethoxysilyl)propyldimethyloctadecyl ammonium chloride (Si-QAC) was included using a concentration of 0.5%. A two-step application process was used, where PNCS microgel was applied to cotton cellulose firstly, followed by the deposition of APTES or Si-QAC secondly. Morphological and chemical changes in the functionalized samples were studied by SEM and FT-IR, and the influence of the polysiloxane matrix structure on functional properties was studied by the determination of the antibacterial activity, moisture content, water vapour transition rate and water uptake. Additionally, for the first time, the cytotoxicity of the samples in the presence of APTES and Si-QAC was assessed. The results show that different structures of the bio-barrier-forming polysiloxane matrices not only have a great influence on functional antimicrobial and responsive properties but also affect the cytotoxicity. Both polysiloxane matrices produced excellent antimicrobial activity, which was also obtained with 0.5% Si-QAC. However, a much higher concentration of APTES (i.e., 4%) was needed for the same antimicrobial effect. Despite its small concentration, the presence of the Si-QAC caused strong cytotoxicity, whereas the other studied samples were non-cytotoxic. The Si-QAC hindered the dual temperature- and pH-responsive properties, but the latter quality was preserved in the presence of APTES.