Pokkah boeng, caused by Fusarium verticillioides, is a serious disease in sugarcane industry. The disease severity is related to the sugarcane genotype as well as environmental considerations, such as nitrogen application. The impact of the nitrogen source (ammonium sulfate, urea, or sodium nitrate) on sugarcane pokkah boeng disease and its pathogen was investigated in planta and fungal growth and sporulation production was measured in vitro. The results showed that ammonium and nitrate were beneficial to fungal mycelium growth, cell densities, and sporulation, which enhanced the disease symptoms of sugarcane pokkah boeng compared to urea fertilization. A total of 1,779 transcripts out of 13,999 annotated genes identified from global transcriptomic analysis were differentially expressed in F. verticillioides CNO-1 grown in the different sources of nitrogen. These were found to be involved in nitrogen metabolism, transport, and assimilation. Many of these genes were also associated with pathogenicity based on the PHI-base database. Several transcription factors were found to be associated with specific biological processes related to nitrogen utilization. Our results further demonstrated that nitrogen availability might play an important role in disease development by increasing fungal cell growth as well as influencing the expression of genes required for successful pathogenesis.