Although no gold standard exists to assess a patient's anticholinergic burden, a review identified 19 anticholinergic burden scales (ABSs). No study has yet evaluated whether a high anticholinergic burden measured with all 19 ABSs is associated with in-hospital mortality and length of stay (LOS). We conducted a cohort study at a Swiss tertiary teaching hospital using patients' electronic health record data from 2015-2018. Included were patients aged ≥65 years, hospitalised ≥48 h without stays and >24 h in intensive care. Patients' cumulative anticholinergic burden score was classified using a binary (<3: low, ≥3: high) and categorical approach (0: no, 0.5-3: low, ≥3: high). In-hospital mortality and LOS were analysed using multivariable logistic and linear regression, respectively. We included 27,092 patients (mean age 78.0 ± 7.5 years, median LOS 6 days). Of them, 913 died. Depending on the evaluated ABS, 1370 to 17,035 patients were exposed to anticholinergics. Patients with a high burden measured by all 19 ABSs were associated with a 1.32- to 3.03-fold increase in in-hospital mortality compared with those with no/low burden. We obtained similar results for LOS. To conclude, discontinuing drugs with anticholinergic properties (score ≥3) at admission might be a targeted intervention to decrease in-hospital mortality and LOS.