To compare the microbiological features in middle meatus samples from chronic rhinosinusitis (CRS) patients with nasal polyps (CRSwNP) and those without nasal polyps (CRSsNP), and control subjects. A total of 136 CRSwNP patients, 66 CRSsNP patients, and 49 control subjects who underwent endoscopic surgery in Beijing TongRen Hospital were enrolled between January 2014 and January 2016. Swab samples were obtained from the middle meatus during surgery and processed for the presence of aerobic and non-aerobic bacteria and fungi. Information on the allergic rhinitis, asthma, the percentage of eosinophils in peripheral blood, and the history of smoking and surgery was collected. The overall isolation rate for bacteria was 81.3% for the three groups, with the lowest in the CRSsNP group (77.3%) and the highest in the CRSwNP group (88.4%). There were no significant differences in isolation rates among the three groups (P = 0.349). The three most common bacterial species were: Coagulase-negative Staphylococcus (24.3%), Corynebacterium (19.9%), and Staphylococcus epidermidis (19.1%) in the CRSwNP group; S. epidermidis (21.2%), Corynebacterium (21.2%), Coagulase-negative staphylococcus (18.2%), and Staphylococcus aureus (13.6%) in the CRSsNP group; S. epidermidis (30.6%), Coagulase-negative Staphylococcus (28.6%), and S. aureus (14.3%) in the control group. For the bacterial species with high isolation rates, no significant difference in the microbial cultures was observed among the three groups; whereas in the CRSwNP group, a relatively high proportion of Citrobacter (5.9%, a bacterium with low isolation rate) was observed compared with the CRSsNP and control groups (all 0.0%). Furthermore, when samples were categorized into subgroups according to the percentage of eosinophils, some bacterial species showed different rates in the CRSwNP group (e.g., S. aureus, 3.3% in the subgroup with normal percentage of eosinophils, 17.2% in the subgroup with increased percentage of eosinophils, P = 0.011). There were no significant differences in the microbiological features (except Citrobacter) in middle meatus samples from CRSwNP patients, CRSsNP patients, and control subjects. S. aureus may promote eosinophilic inflammatory response, while S. epidermidis may promote non-eosinophilic inflammatory response.