Tens of thousands of insects are deposited in collections every year as a result of survey-based studies that aim to investigate ecological questions. DNA-based techniques can expand the utility of these collections to explore their demographic and evolutionary history, temporal changes in their abundance, and pathogen dynamics. Using museum collections of the non-model bee species Eucera (Peponapis) pruinosa Say 1837 (Hymenoptera: Apidae: Eucerini), we developed a standard minimally-destructive and budget-friendly protocol to extract DNA and amplify common gene-fragments for barcoding, phylogenetic analysis, and pathogens. We also generated genome-wide single nucleotide polymorphism (SNP) data from DNA sequencing (ddRADseq) libraries for population structure analyses. We systematically studied the effect of specimen age (≤10 years ago) and tissue type (whole bees vs. abdomen) on DNA quality, single gene-fragment amplification, and SNP calling. We found that all analyses were achievable with both tissue types, yet with variable levels of efficiency because of general DNA degradation. Specifically, we found that not all samples yielded satisfactory results for molecular studies; however, we did not find a systematic effect of specimen age on DNA quality which is encouraging for future studies involving historical specimens. We report the first evidence for the presence of the microsporidian pathogen Nosema spp. in squash bees, opening a window for the study of historical changes in disease pressure in this important agricultural pollinator. Our protocols can be used as a template for the design of future experiments that extract multiple pieces of information using DNA-based methods from insect museum stored specimens.