Chronic benzodiazepine (BZD) use in older adults has been linked to cognitive decline, yet its relationship with amyloid pathology in Alzheimer’s disease (AD) remains unclear. This systematic review aimed to synthesize clinical and preclinical evidence on the effects of BZD exposure on cerebral amyloid accumulation. The review followed PRISMA 2020 guidelines and was registered in PROSPERO (CRD420250604701). Studies were included if they investigated the effects of BZD exposure on amyloid-related outcomes using direct measurements of amyloid burden in humans or experimental models. Reviews, genetic studies, and conference abstracts were excluded. Searches were conducted in PubMed and Web of Science up to March 2024. Risk of bias was assessed using the ROBINS-I tool for observational studies and SYRCLE’s tool for preclinical experiments. Due to methodological heterogeneity, results were synthesized narratively. Eleven studies met inclusion criteria (4 clinical, 7 preclinical). Clinical studies consistently showed lower cortical amyloid burden in chronic BZD users, particularly for short-acting compounds. Preclinical findings were mixed: some benzodiazepines, such as midazolam or 4′-chlorodiazepam, reduced amyloid fibril formation and neurotoxicity, while prolonged lorazepam exposure increased Aβ42 and suppressed translocator protein (TSPO) expression. The certainty of evidence was limited by methodological heterogeneity and moderate risk of bias. Nonetheless, converging findings suggest that BZD effects on amyloid pathology are context-dependent, influenced by compound type and treatment duration. These results challenge the view of BZD as uniformly deleterious in aging and AD. This work was self-funded and conducted as part of a doctoral research project. The review was prospectively registered in PROSPERO.