Chemically modified carbon nanotubes are recognized as effective materials for tackling bacterial infections. In this study, pristine multi-walled carbon nanotubes (p-MWCNTs) were functionalized with nitric acid (f-MWCNTs), followed by thermal treatment at 600 °C, and incorporated into a poly(dimethylsiloxane) (PDMS) matrix. The materials’ textural properties were evaluated, and the roughness and morphology of MWCNT/PDMS composites were assessed using optical profilometry and scanning electron microscopy, respectively. The antibiofilm activity of MWCNT/PDMS surfaces was determined by quantifying culturable Escherichia coli and Staphylococcus aureus after 24 h of biofilm formation. Additionally, the antibacterial mechanisms of MWCNT materials were identified by flow cytometry, and the cytotoxicity of MWCNT/PDMS composites was tested against human kidney (HK-2) cells. The results revealed that the antimicrobial activity of MWCNTs incorporated into a PDMS matrix can be efficiently tailored through nitric acid functionalization, and it can be increased by up to 49% in the absence of surface carboxylic groups in f-MWCNT samples heated at 600 °C and the presence of redox activity of carbonyl groups. MWCNT materials changed the membrane permeability of both Gram-negative and Gram-positive bacteria, while they only induced the production of ROS in Gram-positive bacteria. Furthermore, the synthesized composites did not impact HK-2 cell viability, confirming the biocompatibility of MWCNT composites.