In recent years, metagenomic analyses using next-generation sequencing have advanced the study of indoor microbial communities. This study examined the structure and diversity of bacterial and eukaryotic communities in samples from various Japanese residences, ranging from traditional to modern houses. Microbial sampling was conducted over multiple years during summer and winter in living rooms and bedrooms, targeting diverse surfaces. Simultaneously, indoor environmental parameters—including temperature, relative humidity, and CO2 concentration—were measured. Housing characteristics and resident behaviors, such as building age, pet ownership, and HVAC use, were also recorded. Prior research has linked floor dust microbiomes to asthma and suggested that condensation in air-conditioning units may foster mold growth. Considering humans inhale roughly 15 m3 of air per day, this study focused on microbial communities in indoor/outdoor air, floors, and air conditioner (AC) units. Results showed that relative humidity was the most influential factor shaping microbial communities. In summer, higher humidity (RH > 60%) was associated with increased richness and abundance of eukaryotic microbes, mainly fungi. In winter, higher humidity correlated with increased bacterial evenness and greater proportions of beneficial bacteria such as Lactobacillus. Conversely, under winter heating conditions, ACs operating at higher temperatures harbored greater amounts of pathogenic bacteria including Streptococcus and Haemophilus. These findings suggest that temperature, humidity control, and AC use influence microbial exposure in residential environments.