E-cigarettes are often perceived as safer than cigarettes, but previous research suggests that e-cigarettes can alter respiratory innate immune function. The respiratory microbiome plays a key role in respiratory host defense, but the effect of e-cigarettes on the respiratory microbiome has not been studied. Using 16S rRNA gene sequencing on nasal epithelial lining fluid samples from adult e-cigarette users, smokers, and nonsmokers, followed by novel computational analysis of pairwise log ratios, we determined that e-cigarette use and smoking causes differential respiratory microbiome dysbiosis, which was further affected by sex. We also collected nasal lavage fluid for analysis of immune mediators associated with host-microbiota interactions. Our analysis identified disruption of the relationships between host-microbiota mediators in the nose of e-cigarette users and smokers, which is indicative of disrupted respiratory mucosal immune responses. Our approach provides a novel platform that robustly identifies host immune dysfunction caused by e-cigarette use or smoking.