Mānuka honey, a wound treatment used to eradicate bacteria, resolve inflammation, and promote wound healing, is a current focus in the tissue engineering community as a tissue template additive. However, Manuka honey’s effect on neutrophils during the inflammation-resolving phase has yet to be examined. This study investigates the effect of 0.5% and 3% Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes from a dHL-60 neutrophil model in the presence of anti-inflammatory stimuli (TGF-β, IL-4, IL-4 +IL-13). We hypothesized that Manuka honey would reduce the output of pro-inflammatory signals and increase the release of anti-inflammatory signals. The results of this study indicate that 0.5% honey significantly increases the release of CXCL8/IL-8, CCL2/MCP-1, CCL4/MIP-1β, CCL20/MIP-3α, IL-4, IL-1ra, and FGF-13 while reducing Proteinase 3 release in the anti-inflammatory-stimulated models. However, 3% honey significantly increased the release of TNF-α and CXCL8/IL-8 while reducing the release of all other analytes. We replicated a subset of the most notable findings in primary human neutrophils, and the consistent results indicate that the HL-60 data are relevant to the performance of primary cells. These findings demonstrate the variable effects of Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes of this model of neutrophil anti-inflammatory activity. This study reinforces the importance of tailoring the concentration of Manuka honey in a wound or tissue template to elicit the desired effects during the inflammation-resolving phase of wound healing. Future in vivo investigation should be undertaken to translate these results to a physiologically-relevant wound environment.