Rapeseed (Brassica napus L.) is a crucial oilseed crop globally, facing challenges like stem rot disease caused by Sclerotinia sclerotiorum. This study aimed to enhance fungal detection for effective disease management using molecular techniques. Leaf samples from four Swedish fields were collected, and DNA was extracted using two methods. Polymerase chain reaction (PCR), agarose gel electrophoresis, and nanopore sequencing were employed for S. sclerotiorum detection based on the Internal Transcribed Spacer (ITS) region. Kraken2 and UNITE databases were utilized for bioinformatics analysis. MinION sequencing coupled with barcoding proved effective for species identification. According to the Kraken findings, Ascomycota and Basidiomycota were the dominant phyla detected, with Bipolaris sp. being the most prevalent genus identified by both DNA extraction methods. Regarding the detection of S. sclerotiorum, the results indicated that the silicon membrane-based modified E.Z.N.A Sp Plant DNA Miniprep kit (Omega Bio-Tek), performed better than the modified PureLink™ Microbiome DNA Purification Kit (Invitrogen) in the Kraken 2. The study highlights the potential of molecular tools in correctly identifying fungal pathogens like S. sclerotiorum. Rapid and precise detection enables timely intervention strategies, crucial for disease management in rapeseed cultivation, ultimately contributing to increased crop production and cost-effectiveness for farmers. Integration of nanopore sequencing in field diagnostics holds promise for on-site disease management in agricultural settings.