Grey leaf spot, caused by Magnaporthe oryzae, causes severe damage on perennial ryegrass (Lolium perenne) turf. In this study, the effects of relative humidity (RH, 88 to 100% at 28°C) on infection, colonization and conidiation of M. oryzae on perennial ryegrass were investigated in controlled humidity chambers. Results showed that the RH threshold for successful M. oryzae infection was ≥92% at 28°C. The advancement of infection on the leaf tissue was further examined with a green fluorescent protein (GFP)-tagged M. oryzae strain. No appressorium formation was found when the inoculum was incubated at RH ≤ 88%. Additionally, the GFP-tagged staining provided a rapid method to quantitatively compare the fungal colonization from leaf tissue at different levels of RH. The fluorescence intensity data indicated that the fungal biomass was highest at 100% RH and there was no fluorescence intensity observed at 88% RH or below. Conidiation was only observed when RH was ≥96%, with the most abundant conidiation occurring 8 days after inoculation. Reduced conidiation was associated with decreasing RH, and no conidiation occurred at RH ≤ 92%. This study indicates that infection and conidiation of M. oryzae on perennial ryegrass required different thresholds: 92% and 96% RH for infection and conidiation, respectively. The quantitative data from this research will assist in prediction of grey leaf spot disease outbreaks and of secondary infection of perennial ryegrass.