Human milk oligosaccharides (HMOs) are present in high numbers in milk of lactating women. They are beneficial to gut health and the habitant microbiota, but less is known about their effect on cells from the immune system. In this study, we investigated the direct effect of three structurally different HMOs on human derived macrophages before challenge with (). The study demonstrates that individual HMO structures potently affect the activation, differentiation and development of monocyte-derived macrophages in response to . 6´-Sialyllactose (6'SL) had the most pronounced effect on the immune response against , as illustrated by altered expression of macrophage surface markers, pointing towards an activated M1-like macrophage-phenotype. Similarly, 6'SL increased production of the pro-inflammatory cytokines TNF-α, IL-6, IL-8, IFN-γ and IL-1β, when exposing cells to 6'SL in combination with compared with alone. Interestingly, macrophages treated with 6'SL exhibited an altered proliferation profile and increased the production of the classic M1 transcription factor NF-κB. The HMOs also enhanced macrophage phagocytosis and uptake of . Importantly, the different HMOs did not notably affect macrophage activation and differentiation without exposure. Together, these findings show that HMOs can potently augment the immune response against , without causing inflammatory activation in the absence of , suggesting that HMOs assist the immune system in targeting important pathogens during early infancy.