The particulate and biological components of indoor air have a substantial impact on human health, especially immune respiratory conditions such as asthma. To better explore the relationship between allergens, the microbial community, and the indoor living environment, we sampled the bedrooms of 65 homes in the Chicago area using 23the patient-friendly Inspirotec electrokinetic air sampling device, which collects airborne particles for characterization of both allergens and microbial DNA. The sampling device captured sufficient microbial material to enable 16S rRNA amplicon sequencing data to be generated for every sample in the study. Neither the presence of HEPA filters nor the height at which the air sampling device was placed had any influence on the microbial community profile. A core microbiota of 31 OTUs was present in more than three quarters of the samples, comprising around 45% of the relative sequence counts in each bedroom. The most abundant single organisms were Staphylococcus, with other core taxa both human and outdoor-associated. Bacterial alpha diversity was significantly increased in bedrooms that reported having open windows, those with flowering plants in the vicinity, and those in homes occupied by dogs. Porphyromonas, Moraxella, Sutterella, and Clostridium, along with family Neisseraceae, were significantly enriched in homes with dogs; interestingly, cats did not show a significant impact on microbial diversity or relative abundance. While dog allergen load was significantly correlated with bacterial alpha diversity, the taxa that significantly correlated with allergen burden did not exclusively overlap with those enriched in homes with dogs. Alternaria allergen load was positively correlated with bacterial alpha diversity, while Aspergillus allergen load was negatively correlated. The Alternaria allergen load was also significantly correlated with open windows. Microbial communities were significantly differentiated between rural, suburban, and urban homes and houses that were physically closer to each other maintained significantly more similar microbiota. We have demonstrated that it is possible to determine significant associations between allergen burden and the microbiota in air from the same sample and that these associations relate to the characteristics of the home and neighborhoods.