Aims: Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest. Methods and Results: Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25°C, indicating the secretion of active enzyme by the fungus, and one-third produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15°C and some at 39°C. Conclusions: Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates. Significance and Impact of the Study: To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities.