Postbloom fruit drop (PFD) of citrus and Key lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indole-acetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection.