An assessment of the opportunistic microbiota of dust in the “air–phylloplane-sealed surfaces” system was conducted in various functional zones of the urban ecosystem, using Moscow as an example during the summer and autumn, corresponding to the early and late phases of the trees’ vegetative season. Three distinct functional zones were selected for the study, each representing varying levels of anthropogenic impact: a high-traffic zone, a residential zone, and a recreational zone. The concentration of microfungi ranged from 590 to 660 CFU/m3 in the air, 8 × 103 to 34 × 103 CFU/g on leaf surfaces, and 7 × 103 to 25 × 103 CFU/g in road dust during the summer. In autumn, the concentrations were 35–98 CFU/m3, 28 × 103 to 64 × 103 CFU/g, and 11 × 103 to 25 × 103 CFU/g, respectively. The concentration of opportunistic fungi in the air decreased by up to 6 times in autumn and by up to 2 times in road dust across all functional zones, while increasing up to 4 times on leaf surfaces in the residential and high-traffic zones. The largest proportion of BSL-2 (potential agents of opportunistic mycoses) fungi was recorded in the traffic zone at the end of vegetative season, comprising 89%. Regardless of substrate type or functional zone, fungi of the genus Aspergillus, which are toxic to humans, predominated, with A. niger showing the highest abundance in all functional zones. Each substrate was also characterized by different dominant species: Cladosporium cladosporioides in the air, Penicillium aurantiogriseum and Hyphopichia burtonii on leaf surfaces, and A. fumigatus and A. niger in road dust. The proportional participation of opportunistic fungal species identified both at the beginning and the end of the trees’ growing season was 40% for air and road dust, and 38.5% for leaf surfaces, which suggests that this component of the cultivable mycobiota remains stable across the studied habitats.