The fungal colonisation of indoor and outdoor surfaces not only damages the aesthetic but may also be a source of infection for immunocompromised and/or sensitive individuals. We used macro attenuated total reflection-Fourier transform infrared (macro ATR-FTIR) microspectroscopy and scanning electron microscopy (SEM) to assess the adhesion pattern of conidia from Aspergillus brasiliensis ATCC 9642 and Aureobasidium pullulans ATCC 15233 on polyester surfaces. The surfaces were completely covered by Aureobasidium pullulans germinated conidia after 7 days, while Aspergillus brasiliensis conidia were poorly attached and were not able to germinate in the same period. Carbohydrates and polysaccharides were observed in polyester and control surfaces suggesting their major role on the Aspergillus brasiliensis conidia attachment. Thus, synchrotron macro ATR-FTIR technique is confirmed as a powerful molecular characterization tool to investigate surface distribution of the key chemical compounds during fungal adhesion at high resolution.