<p>The synthesis route and matrix material source play a crucial role in determining the characteristics and bioactivity of a composite. This study focuses on synthesizing a PANI/Silica-Coated Magnetite composite with variations in synthesis methods and silica sources. The in situ method involves polymerizing polyaniline within a silica gel containing magnetite, while the ex situ method incorporates pre-formed polyaniline into a silica gel through a sol-gel condensation reaction. Two silica sources, Na₂SiO₃ and Tetraethyl Orthosilicate (TEOS), were utilized. FTIR analysis confirmed the successful polymerization of PANI, as evidenced by an absorption peak at 1110 cm⁻¹, indicating the vibration of C-N bonds attached to aromatic amines. Vibrating Sample Magnetometer (VSM) analysis demonstrated that the PANI composite containing magnetite exhibited magnetic properties, unlike pure PANI. Additionally, TGA characterization revealed that the composite possessed enhanced thermal stability compared to pure PANI. Bioactivity tests against E. <i>coli</i>, S. <i>aureus</i>, and C. <i>albicans</i> showed that, overall, composites synthesized using the in situ method exhibited superior bioactivity compared to those synthesized via the ex situ method. Among them, the In Situ PANI/TEOS-Magnetite (IPTM) composite showed the most effective antibacterial and antifungal activity.</p> Graphical Abstract <p></p>