In order to identify the secondary metabolism-related genes of Phanerochaete chrysosporium growing under pure O2 and nitrogen-limited conditions, 2322 ESTs fragments originated from two suppression-subtractive libraries were analyzed using the cDNA microarray technique. Ten significantly upregulated and 22 significantly downregulated genes were identified in the 72 h cultured mycelia RNA samples (secondary metabolism). According to qPCR, 16 out of the 32 genes were expressed differently in secondary metabolism. Transcripts of secondary metabolism up-regulation genes exhibited homologies to aryl-alcohol dehydrogenase (SSh1554), ABC transporter gene (SSH624), chitinase (SSH963), heat shock protein (SSH1193), catalase (SSH317), cytochrome P450 (SSH331), glucosamine-6-phosphate isomerase (SSH611), and alkyl hydroperoxide reductase (SSH362) genes. Ninety-three genes could be classified by Eukaryotic Orthologous Groups (KOG). Among the genes assigned a function, gene expression patterns were different in both secondary metabolism and primary metabolism. In the group of “Cellular Processes and Signaling,” most of the genes were from the primary metabolism library. On the other hand, genes from the secondary metabolism library were found mainly in the “Information Storage” and “Processing and Poorly Characterized” groups. Based on the KOG functional assignments, six genes belong to the ubiquitin system, and all of them were from primary metabolism phase. The presence of the H2O2-relevant genes suggested that parts of the genes expressed in 72 h might be involved in the ligninolytic process during secondary metabolism of P. chrysosporium.