We demonstrate a new sensitive biosensor for detection of vital fungal spores of Aspergillus niger. The biosensor is based on silicon microfabricated cantilever arrays operated in dynamic mode. The change in resonance frequency of the sensor is a function of mass binding to the cantilever surface. For specific A. niger spore immobilization on the cantilever, each cantilever was individually coated with anti-Aspergillus niger polyclonal antibodies. We demonstrate the detection of single A. niger spores and their subsequent growth on the functionalized cantilever surface by online measurements of resonance frequency shifts. The new biosensor operating in humid air allows quantitative and qualitative detection of A. niger spores as well as detection of vital, functional spores in situ within ∼4 h. The detection limit of the sensor is 103 CFU mL−1. Mass sensitivity of the cantilever sensor is ∼53 pg Hz−1.