White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 time higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.