Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.