Bio-based, renewable and biodegradable products with multifunctional properties are also becoming basic trends in the textile sector. In this frame, cellulose nanofibrils (CNFs) have been surface modified with hexamethylenediamine/HMDA and used as an antimicrobial additive to a ring-spun viscose yarn. The CNF-HMDA suspension was first characterized in relation to its skin irritation potential, antimicrobial properties, and technical performance (dispersability and suspensability in different media) to optimize its sprayability on a viscose fiber sliver with the lowest sticking, thus to enable its spinning without flowing and tearing problems. The impact of CNF-HMDA content has been examined on the yarn`s fineness, tensile strength, surface chemistry, wettability and antimicrobial properties. The yarn`s antimicrobial properties were increasing with the content of CNF-HMDA, given a 99% reduction for S. aureus and C. albicans (log 1.6–2.1) in up to 3 h of exposure at minimum 33 mg/g, and for E. coli (log 0.69–2.95) at 100 mg/g of its addition, yielding 45–21% of bactericidal efficacy. Such an effect is related to homogeneously distributed CNF-HMDA when sprayed from a fast-evaporated bi-polar medium and using small (0.4 mm) nozzle opennings, thus giving a high positive charge (0.663 mmol/g) without affecting the yarn`s tenacity and fineness, but improving its wettability. However, a non-ionic surfactant being used in the durability testing of functionalized yarn to 10-washing cycles, adheres onto it hydrophobically via the methylene chain of the HMDA, thus blocking its amino groups, and, as such, decreasing its antibacterial efficiency, which was slightly affected in the case when the washing was carried out without using it.