BackgroundStill, little is known about microbial dysbiosis in oropharyngeal and laryngeal tissue as risk factor for development of local squamous cell carcinoma. The site‐specific microbiota at these regions in healthy and cancer tissue and their modulation by environmental factors need to be defined.MethodsThe local microbiota of cancer tissue and healthy controls was profiled by 16S rRNA gene amplicon sequencing and statistical analysis using 111 oropharyngeal and 72 laryngeal intraoperative swabs.ResultsOropharynx and larynx harbor distinct microbial communities. Clear effects of both smoking and cancer were seen in the oropharynx whereas effects in the larynx were minor.ConclusionThe distinct microbial communities at larynx and oropharynx partially explain why the effects of cancer and smoking were distinct at those sites. Thus, the use of microbiota supposed to mirror community changes in another target location should be avoided and more studies on the actual cancerous environment are necessary.