Alzheimer’s disease (AD) is a neurodegenerative disorder that primarily affects individuals over 60 years of age, characterized by symptoms such as memory impairment and cognitive decline. The pathogenesis of AD involves multiple factors, including protein misfolding and oxidative stress. A crucial aspect of AD progression is the dysregulation of cholinesterase enzymes, particularly acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which contribute to neurotoxic amyloid plaques and neurofibrillary tangles. This study investigates the potential of proteins and peptides from the venom of Calloselasma rhodostoma as BChE inhibitors, aiming to explore new therapeutic avenues for AD. Venom was extracted, fractionated, and analyzed using ultrafiltration, SDS-PAGE, and LC-HRMS. In vitro assays evaluated the BChE inhibition activity, while in silico molecular docking assessed the binding affinities of the identified peptides. The study identified several venom-derived peptides with significant BChE inhibitory potential, notably CFVVQPWEGK and IDVLSDEPR, which demonstrated strong binding affinities and stability in docking studies. These findings highlight the potential of peptides derived from C. rhodostoma venom as natural BChE inhibitors, offering a promising basis for developing novel AD therapies. Further research is warranted to fully understand the mechanisms and therapeutic potential of these bioactive compounds.