The development of antimicrobial fabric with comfort ability for biomedical applications in a scalable, cost-effective coating process is now extremely challenging. This study aims to develop an antimicrobial comfortable single jersey knit fabric with in-situ generated AgNPs (silver nanoparticles) adhesion in a facile technique for biomedical applications (e.g., wound dressing). The weft knitted fabrics of cotton and blends with polyester in different proportion were independently treated with 5% AgNO3 solutions by mechanical thermo-fixation technique using the pad-dry-cure procedure under optimal conditions on 60/40 CVC (Chief value to cotton), 100% cotton and 65/35 PC (polyester/cotton) fabrics. Then microbial assay, Fourier transforms infrared spectroscopy (FTIR), moisture management test (MMT), and fabric touch Test (FTT) were employed to assess the antimicrobial, bonding, moisture, and comfort aspects. The treated fabrics show consistent antimicrobial properties without any significant differences, the Zone of Inhibition (ZOI) against S. aureus measures 11 ± 1.4 mm for 60/40 CVC @T, while 65/35 PC @T and 100% Cotton @T exhibit ZOI of 10 ± 2.7 mm and 9 ± 1.9 mm, respectively. Furthermore, treating the fabrics with AgNO3 enhances their ability to manage moisture, facilitating faster absorption and quicker drying compared to untreated fabrics where 100% Cotton @T show maximum OMMC (overall moisture management characteristics) value of 0.371 ± 0.012. Fourier transforms infrared spectroscopy analysis verifies the absence of bonding between metal silver ions and fiber molecules, potentially as a result of metal silver ions being adsorbed. Moreover, metal silver ions fabrics have greater overall moisture management capabilities than untreated counterparts, with 60/40 CVC fabric outperforming 100% cotton and 65/35 PC (polyester/cotton) fabrics. In conclusion, FTT analysis indicates that the presence of metal silver ions has negligible effects on fabric comfort. However, 100% Cotton @T fabric stands out for its superior comfort properties such as the smoothness, softness, and warmth indices show scores by 0.34, 0.87, and 0.72 respectively compared to other treated fabrics. Nevertheless, the treated fabrics receive high scores for attributes like smoothness, softness, and warmth. The use of AgNO3 treatment suggests that fabrics could be suitable for biomedical applications without hindering fabrics comfortability.