Antimicrobial textiles are helpful tools to fight against multidrug-resistant pathogens and nosocomial infections. The deposition of silver nanoparticles (AgNPs) onto textiles has been studied to achieve antimicrobial properties. Yet, due to health and environmental safety concerns associated with such formulations, processing optimizations have been introduced: biocompatible materials, environmentally friendly agents, and delivery platforms that ensure a controlled release. In particular, the functionalization of polyester (PES) fabric with antimicrobial agents is a formulation in high demand in medical textiles. However, the lack of functional groups on PES fabric hinders the development of cost-effective, durable systems that allow a controlled release of antimicrobial agents. In this work, PES fabric was functionalized with AgNPs using one or two biocompatible layers of chitosan or hexamethyldisiloxane (HMDSO). The addition of organo-matrices stabilized the AgNPs onto the fabrics, protected AgNPs from further oxidation, and controlled their release. In addition, the layered samples were efficient against Staphylococcus aureus and Escherichia coli. The sample with two layers of chitosan showed the highest efficacy against S. aureus (log reduction of 2.15 ± 1.08 after 3 h of contact). Against E. coli, the sample with two layers of chitosan showed the best properties. Chitosan allowed to control the antimicrobial activity of AgNPs, avoid the complete loss of AgNPs after washings and act in synergy with AgNPs. After 3 h of incubation, this sample presented a log reduction of 4.81, and 7.27 of log reduction after 5 h of incubation. The antimicrobial results after washing showed a log reduction of 3.47 and 4.88 after 3 h and 5 h of contact, respectively. Furthermore, the sample with a final layer of HMDSO also presented a controlled antimicrobial effect. The antimicrobial effect was slower than the sample with just an initial layer of HMDSO, with a log reduction of 4.40 after 3 h of incubation (instead of 7.22) and 7.27 after 5 h. The biocompatibility of the composites was confirmed through the evaluation of their cytotoxicity towards HaCaT cells (cells viability > 96% in all samples). Therefore, the produced nanocomposites could have interesting applications in medical textiles once they present controlled antimicrobial properties, high biocompatibility and avoid the complete release of AgNPs to the environment.