Fungal infection causes a significant reduction in both quality and yield in different crop plants. Invasive fungal species cause serious community and health care-acquired infections worldwide. It is essential to precisely detect and identify the pathogenic fungi in the seeds and/or at an early stage of plant growth to reduce the yield losses of cultivated crops. Conventional laboratory techniques, for example, microscopic and cultural techniques, are labor-intensive, time-consuming, and involve complicated sample handling. Methods involved in the diagnosis of fungal diseases include visual inspection of symptoms, serological assays, and DNA-based detection of pathogenic fungi. But most of these techniques are time-consuming and costly, provide erroneous results from cross-contamination, and require expert laboratory personnel. Another significant disadvantage is their inability to detect pathogens on-site in field conditions. In contrast, a combination of immunological and molecular diagnostics with nanotechnology-based strategies offers rapid, convenient, cheap, specific, and accurate detection of pathogenic fungi. The functionalized nanoparticles can be used to develop pathogen detection devices with smart sensing capabilities for both laboratory and field applications. This chapter provides a comprehensive overview of the recent advances in nanotechnology-based strategies for the detection of pathogenic fungi.