Chronic rhinosinusitis is an inflammatory condition that affects up to 12% of people in developed countries. Previous studies examining the association of the sinus bacterial microbiota with CRS have found inconsistent results, possibly because of inconsistencies in sampling site. In this study we aimed to determine if the sinus microbiota is altered in CRS compared to controls, and additionally if the middle meatus is a representative site for sampling. Swab samples were collected from 12 healthy controls and 21 CRS patients, including all eight sinuses for CRS patients, and between one and five sinuses for controls. Middle meatus and nostril swabs were also collected. Samples were subject to 16S rRNA gene sequencing, which revealed significant differences in sinus microbiomes between CRS and controls. The Enterobacteriaceae were over-represented in CRS, while the Alloiococcus and Corynebacterium genera were more abundant in healthy sinuses. Comparisons of the middle meatus between groups did not reveal these differences, and underestimated the abundance of the Enterobacteriaceae. Additionally, intra-patient variation was lower betweens sinuses than between sinus and middle meatus, suggesting that this sinuses as possible and performing 16S rRNA gene sequencing.