NK cells participate in the antimicrobial immune response. NK cell activation by bacterial polysaccharides (PSs) can occur indirectly through signals from other immune cells or through direct influence of PSs on NK cells via interaction with activating receptors. This study demonstrated that peripheral NK cells bind fluorescently labeled PSs from the Gram-negative bacteria Escherichia coli, Shigella flexneri, and Pseudomonas aeruginosa with varying efficiency depending on the PS origin and the NK cell donor. PSs from Escherichia coli strain O19ab induced an increased IFN-γ production by NK cells in the absence of high levels of PS binding to the cell surface. The obtained results provide new insights into the interactions of NK cells with polysaccharides and open new prospects for understanding the NK cell-mediated immune response to bacterial infections.