Chitosan is an environmentally friendly agent that is used to achieve the antimicrobial properties of textiles. Nowadays, the binding of chitosan to the textiles has been thoroughly researched due to the increasing demands on the stability of achieved properties during the textile care processes. Most crosslinking agents for chitosan are not safe for humans or environment, such as glutaric aldehyde (GA) and formaldehyde derivatives. Eco-friendly polycarboxyilic acids (PCAs) are usually used in after-treatment. In this work, chitosan powder was dissolved in citric acid with sodium hydrophosphite (SHP) as a catalyst. Standard cotton (CO) and polyester/cotton (PES/CO) fabrics were pretreated in 20% NaOH, similar to mercerization, in order to open the structure of the cotton fibers and hydrolyze polyester fibers, continued by finishing in the gelatin chitosan bath. Afterwards, the hot rinsing process, followed by drying and curing, closed the achieved structure. The main objective was to achieve durable antimicrobial properties to multiple maintenance cycles CO and PES/CO fabric in order to apply it in a hospital environment. The characterization of fabrics was performed after treatment, first and fifth washing cycles according ISO 6330:2012 by field emission scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FTIR-ATR), electrokinetic analysis (EKA), by the determination of tensile properties and mechanical damage (wear), and the antimicrobial activity. The application of 20% NaOH led to the swelling and mercerization of cotton cellulose, and hydrolysis of polyester, resulting in better mechanical properties. It has been confirmed that the chitosan particles were well implemented into the cotton fiber and onto to the polyester component of PES/CO blend. The presence of chitosan was confirmed after five washing cycles, but in lower quantity. However, achieved antimicrobial activity is persistent.