In order to impart barrier properties against water and microorganisms on breathable three dimensional spacer fabrics as medical or technical textiles, fabric samples were treated with two water repellent agents and a quaternary ammonium salt namely cetyltrimethylammonium bromide (CTAB), using pad-dry-cure method. Two different water repellent agents based on hydrocarbon and acrylic copolymer were used. The water repellent property of samples was tested by Bundesmann and contact angle tests. Antimicrobial activity of samples was analyzed quantitatively according to AATCC 100. Simultaneous finishing of samples was done with 3% CTAB and 4% fluoroalkyl acrylic copolymer. To study the effect of various treatments on comfort related properties, air and water vapor permeability, water repellency and compression were measured. The results showed that the antimicrobial and water repellent spacer fabrics can be achieved applying selected material without significant changes on their comfort properties. Also a regression model was presented to predict the water vapour permeability of knitted spacer fabrics based on course density (CPC) changing.