Airborne fungal spores of the genus Alternaria pose challenges for accurate airborne spore identification by automatic bioaerosol monitors, because of their significant implications for public health and agriculture due to their role as airborne allergen and plant pathogen. These systems require high-quality reference data for training algorithms by machine learning. Alternaria alternata was cultured on three different media, including exposure to UV light to favor sporulation. Spore morphology was evaluated both macroscopically and microscopically, and chemical analysis was conducted using micro-Raman spectroscopy to assess spore composition. Significant differences were observed in colony morphology and spore characteristics among culture media. While typical spores predominated, atypical forms were also identified, which may represent a potential bias for identification. Comparative analysis with air samples by the Hirst method also revealed overall differences in spore morphology pattern. Standardizing culture conditions and accounting for variability in spore properties are essential for improving the reliability of bioaerosol monitoring systems. Further research is needed to refine detection methods for A. alternata and other airborne fungal spores.