Neutrophils are a major source of reactive oxygen species (ROS). The role of airway mucous on ROS production is unknown. The aim of our study was to investigate the direct influence of bronchoalveolar lavage fluid (BALF) and induced sputum (IS) alone or in combination with chemical/biological stimulus on ROS production in peripheral blood neutrophils during chronic obstructive pulmonary disease (COPD). Neutrophils were isolated from peripheral blood of 47 patients with moderate COPD and 14 healthy individuals (HI). BALF/RPMI (1:1) or IS/RPMI (1:1) from COPD patients were used to stimulate neutrophils alone or in combination with phorbolmyristate- acetate (PMA) (0.1–30 nM) or Staphylococcus aureus bacteria (0.7–500 bact/neutrophil). Relative generation of ROS was measured flow cytometrically. BALF/RPMI and in combination with relatively low PMA or all bacteria concentrations stimulated ROS; while, combination with relatively high PMA concentrations suppressed ROS in of COPD patients and HI. IS/RPMI and its combination with PMA inhibited ROS generation in both groups; whereas, IS stimulated or had a tendency to stimulate ROS production with relatively high bacteria concentrations. In conclusion, BALF and IS directly or in combination with chemical/biological factors modulated ROS production. This effect was stronger in neutrophils from COPD patients and depended on chemical/biological stimulus intensity.