Over the years, secondary metabolites have proven to be significant in the discovery of novel antibiotics and/or lead compounds. Various secondary metabolites have been reported to be produced by fungus of the genus, Alternaria. Here we report the isolation of secondary metabolites by the bioassay-guided fractionation of the ethyl acetate extract of fermentation cultures of the fungus, Alternaria tenuissima. This fungal strain was isolated from the soil underlying the lichen, Peltigera didactyla; and the lichen was collected from Wapusk National Park, a location in Northern Manitoba. The compounds isolated from the fungus include deoxyphomalone, dimethyl 4-methyl-2,6-pyridinedicarboxylate, stemphyperylenol and N-Methyl pyrrolidinone. Their structures were determined by comprehensive analysis of their spectroscopic data including FT-IR, mass spectrometry, 1D and 2D NMR; and their bioactivities were tested against E. coli cells. Some of the compounds demonstrated some bioactivity. The taxonomic identity of the fungus was confirmed by ITS sequencing of its ribosomal DNA.