Background: There is increasing evidence that nitric oxide (nitrogen monoxide, NO) significantly influences immune cellular responses, including those from polymorphonuclear leukocytes (PMNs). Objective: The aim of this study was to examine a possible effect of NO on PMNs’ function (chemotaxis, production of reactive oxygen species (ROS), and NETosis) using live cell imaging. Moreover, we investigated PMN surface epitope and neutrophil oxidative burst under the influence of NO by flow cytometric analysis. Methods: Whole blood samples were obtained from healthy volunteers, and PMNs were isolated by density centrifugation. Live cell imaging using type I collagen matrix in µSlide IBIDI chemotaxis chambers was conducted in order to observe N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP)-stimulated PMN chemotaxis, ROS production, and NETosis. In the test group, NO was continuously redirected into the climate chamber of the microscope, so the chemotaxis chambers were surrounded by NO. The same experimental setup without NO served as a control. In addition, isolated PMNs were incubated with nitrogen monoxide (NO) or without (the control). Subsequently, flow cytometry was used to analyze neutrophil antigen expression and oxidative burst. Results: Our live cell imaging results demonstrated a migration-promoting effect of NO on PMNs. We observed that in the case of prior stimulation by fMLP, NO has no effect on the time course of neutrophil ROS production and NET release. However, flow cytometric analyses demonstrated an increase in ROS production after pretreatment with NO. No NO-dependent differences for the expression of CD11b, CD62L, or CD66b could be observed. Conclusions: We were able to demonstrate a distinct effect of NO on PMNs’ function. The complex interaction between NO and PMNs remains a major research focus, as the exact mechanisms and additional influencing factors remain elusive. Future studies should explore how varying NO concentrations and the timing of NO exposure relative to PMN activation affect its influence.