Gold nanoparticles of 2–5 nm supported on woven fabrics of activated carbon fibers (ACF) were effective during CO oxidation at room temperature. To obtain a high metal dispersion, Au was deposited on ACF from aqueous solution of ethylenediamine complex [Au(en)2]Cl3 via ion exchange with protons of surface functional groups. The temperature-programmed decomposition method showed the presence of two main types of functional groups on the ACF surface: the first type was associated with carboxylic groups easily decomposing to CO2 and the second one corresponded to more stable phenolic groups decomposing to CO. The concentration and the nature of surface functional groups was controlled using HNO3 pretreatment followed by either calcination in He (300–1273 K) or by iron oxide deposition. The phenolic groups are able to attach Au3+ ions, leading to the formation of small Au nanoparticles (<5 nm) after reduction by H2. This was confirmed by high-resolution electron microscopy combined with X-ray energy-dispersive analysis. The catalyst with high Au dispersion demonstrated high activity in CO oxidation. The surface carboxylic groups decomposed during interaction with [Au(en)2]Cl3 solution and reduced Au3+ to Au0, resulting in the formation of bigger (>9 nm) Au agglomerates after reduction by H2. These catalysts demonstrated lower activity as compared to the ones containing mostly small Au nanoparticles. Complete removal of surface functional groups rendered an inert support that would not interact with the Au precursor. The oxidation state of gold in the Au/ACF catalysts was controlled by X-ray photoelectron spectroscopy before and after the reduction in H2. The high-temperature reduction in H2 (673–773 K) was necessary to activate the catalyst, indicating that metallic gold nanoparticles are active during catalytic CO oxidation.