The present study aims to explore the potential application of proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for real-time monitoring of microbial volatile organic compounds (MVOCs). This investigation can be broadly divided into two parts. First, a selection of 14 MVOCs was made based on previous research that characterized the MVOC emissions of Trichoderma atroviride, which is a filamentous fungus widely used as a biocontrol agent. The analysis of gas-phase standards using PTR-ToF-MS allowed for the categorization of these 14 MVOCs into two groups: the first group primarily undergoes nondissociative proton transfer, resulting in the formation of protonated parent ions, while the second group mainly undergoes dissociative proton transfer, leading to the formation of fragment ions. In the second part of this investigation, the emission of MVOCs from samples of T. atroviride was continuously monitored over a period of five days using PTR-ToF-MS. This also included the first quantitative online analysis of 6-amyl-α-pyrone (6-PP), a key MVOC emitted by T. atroviride. The 6-PP emissions of T. atroviride cultures were characterized by a gradual increase over the first two days of cultivation, reaching a plateau-like maximum with volume mixing ratios exceeding 600 ppbv on days three and four. This was followed by a marked decrease, where the 6-PP volume mixing ratios plummeted to below 50 ppbv on day five. This observed sudden decrease in 6-PP emissions coincided with the start of sporulation of the T. atroviride cultures as well as increasing intensities of product ions associated with 1-octen-3-ol and 3-octanone, whereas both these MVOCs were previously associated with sporulation in T. atroviride. The study also presents the observations and discussion of further MVOC emissions from the T. atroviride samples and concludes with a critical assessment of the possible applications and limitations of PTR-ToF-MS for the online monitoring of MVOCs from biological samples in real time.