Air conditioning is a crucial participant in indoor air circulation and harmful microorganism transmission. To elucidate microbial contamination in air conditioning systems, bacteria, fungi, and pollens in residential air conditioning (AC) filters in Shanghai were revealed. The concentrations of fungal ITS (1,972,037 ± 5,025,505 copies/cm 2 ) in most filter samples were considerably higher than the 16S rRNA gene (21,587 ± 21,461 copies/cm 2 ). The multitudinous bacteria determined a higher richness, whereas the trend was not in line with fungi, owing to Aspergillus flourishing, with an average of 43.75%. Numerous bacteria that may often be prevalent in human skin, mucous membranes, and intestines, such as Enhydrobacter, Micrococcus, Staphylococcus, Streptococcus, and Haemophilus, were also discovered in AC filters. The pollens were affiliated with Streptophyta at phylum, and Humulus was the most abundant genus (mean value 67.84%), raising the possibility of allergic responses. This study provides a better understanding of the microbial characteristics of AC filters, enables more rational approaches for microbe contaminant control, and ultimately improves human health in megacities.