Azoxyglycosides, glycosides of methylazoxymethanol (MAM), are unique to Cycadales plants, with cycasin (β-glucoside) and macrozamin (β-primeveroside) as major components and neocycasins as minor ones. Accurate quantification requires inactivating co-existing β-glycosidases during extraction to prevent hydrolysis, but previous estimates of cycasin and macrozamin contents in seeds were insufficiently cautious. Additionally, a β-primeverosidase that hydrolyzes macrozamin was found in Japanese cycad (Cycas revoluta Thunb.) leaves, with its distribution and activity poorly understood, necessitating similar caution for macrozamin measurement. In this study, we quantified azoxyglycosides in seeds of several cycad species (Cycas media, C. revoluta, Dioon species, D. spinulosum, Ceratozamia mexicana, Macrozamia communis, M. reidlei) and various parts of C. revoluta (leaves, pith, tuberous roots, seedlings, seeds) using freezing-low temperature extraction (80% aqueous ethanol) and high performance liquid chromatography (HPLC) for MAM and azoxyglycosides. We also analyzed glycosidase activities affecting azoxyglycoside estimation. Results showed exceptionally high glucosidase activity toward cycasin in C. media seeds and very low glycosidase activities in M. reidlei seeds; primeverosidase activity was present in all cycad seeds, highest in Dioon species. Azoxyglycoside contents varied: C. revoluta seeds contained 0.9~1.3% cycasin and nearly no macrozamin; C. media seeds had 0.79% cycasin and 1.36% macrozamin; Dioon species seeds had 0.24% cycasin and 0.80% macrozamin; Macrozamia species seeds had 0.19~0.40% cycasin and 1.65~1.77% macrozamin. C. revoluta parts showed distinct azoxyglycoside distributions (e.g., leaves: 0.24~2.00% cycasin, 0~0.38% macrozamin; pith: 0.70~1.00% cycasin, 0.06% macrozamin). Freezing-low temperature extraction minimized MAM formation (except in C. media due to high glucosidase activity), while room-temperature extraction with 95% ethanol resulted in low cycasin recovery (20~40%) for C. revoluta seeds. MAM measurement is crucial for estimating azoxyglycoside contents in conjunction with glycosidase activities. This study first evaluated azoxyglycoside contents in Ceratozamia seeds, various C. revoluta parts, and Dioon macrozamin contents.