Whilst fungi are a large fraction of primary biological aerosol particles (PBAPs) and their impact on global climate has been widely recognised, few studies have empirically assessed fungal vertical profiles and diversity relating those with rainfall. Here, we show the results of fungal PBAPs before and after a rainfall event during a fieldwork campaign using a hot-air balloon over a mixed land-use context at the Brazilian Atlantic Forest biodiversity hotspot. Four flights of c. 1 hour each were performed in the early morning from 8th until 11th of March 2022, and data were collected at three sampling heights (0, 150 and 300 m). Rainfall estimation using IMERG data indicated the precipitation event was of 15–20 mm and ERA5/ECMWF data highlighted that most of the airborne samples were taken above the boundary layer height. After the rainfall, the concentration of fungal spores at the ground level remained unchanged, whereas it was reduced to between 2- and 2.5-fold for the 150 and the 300 m heights, respectively. This was also accompanied by a reduction in the number of Pink-CFU, indicating a major drop in fungal PBAPs at higher altitudes associated with the rain. In addition, total spore concentration indicated Cladosporium sp. as dominant at all sampling heights, accounting for more than 80% of all spores, whereas Aspergillus/Penicillium-like represented less than 20%. Our results show the effects of rainfall and altitude on the concentration of fungal PBAPs, indicating how wet removal impacts fungi vertical profiles which has knock-on-effects on cloud and precipitation formation.