The economically important trunk disease pathogen Phaeomoniella chlamydospora causes Petri disease in Vitis vinifera and is also associated with the Esca trunk disease complex. Not much is known about the pathogen’s epidemiology and interactions with the grapevine host, other trunk disease pathogens and biological control agents such as Trichoderma harzianum. Reporter gene labelling of plant pathogens and biocontrol agents can facilitate host–pathogen–biocontrol interaction studies. Therefore, the first aim of this study was to stably transform T. harzianum strain T77 and a South African isolate of P. chlamydospora with the Green fluorescent protein (GFP) and the Red fluorescent protein (DsRed-Express) reporter genes. Several stably transformed reporter gene labelled transformants were obtained for both fungi. The second aim was to evaluate these transformants and their respective wild type isolates for several in vitro biological characteristics, virulence (P. chlamydospora) and host colonisation (T. harzianum) on hydroponically grown grapevine cuttings. Additionally, a previously described New Zealand P. chlamydospora GFP transformant (pCT74-P7) and its wild type isolate were also biologically characterised. In vitro biological and virulence characteristics of P. chlamydospora transformants did not vary much from their respective wild type isolates. In vitro biological characterisation of T. harzianum transformants showed that one of the transformants exhibited a faster growth rate at lower temperatures. Nonetheless, characterisation of its host colonisation ability on grapevine cuttings showed no difference when compared with another transformant and the wild type isolate. All Trichoderma isolates showed differential colonisation of grapevine cultivars Chenin Blanc and Merlot.