The aim of this study was to characterize the properties of lactoferrin (LF) obtained in a process developed for its isolation from acid whey derived from the production of fresh curd cheese, using a unique technology of ion-exchange chromatography on CIM® monolithic columns. The freeze-dried lactoferrin samples produced on the pilot plant (capacity 1 m3) were examined for the purity, iron-binding capacity, antibacterial activity, and pH- and temperature-stability. Apo-LF inhibited several tested strains (enterobacteria, Staphylococcus, Streptococcus salivarius) except clostridia, lactic acid bacteria, and bifidobacteria. Sample of LF intentionally saturated with Fe3+ lost its antibacterial activity, indicating the involvement of mechanisms based on depriving bacteria of an iron source. All samples, regardless of the iron-saturation level, exhibited stability in pH range 4.0 to 11.0. LF with higher iron content (A-value = 41.9%) showed better thermal stability. Heat treatment up to 72 °C/3 s did not reduce antimicrobial activity against E. coli O157: H7 tox-. Higher purity (above 91%), higher iron-binding capacity and higher inhibitory activity against E. coli O157: H7 tox- compared to some similar products from the market was observed. These results demonstrate a high potential of monolithic ion-exchange chromatography for industrial processing of acid whey as a source of LF that can be used in new products with high-added value. The upscaling of the process is ongoing on a demonstration plant (10−30 m3/day capacity).