Background Echocardiographic quantification of ejection fraction (EF) by manual endocardial tracing requires training, is time-consuming and potentially user-dependent, whereas determination of cardiac output by pulmonary artery catheterization (PAC) is invasive and carries a risk of complications. Recently, a novel software for semi-automated EF and CO assessment (AutoEF) using transthoracic echocardiography (TTE) has been introduced. We hypothesized that AutoEF would provide EF values different from those obtained by the modified Simpson’s method in transoesophageal echocardiography (TOE) and that AutoEF CO measurements would not agree with those obtained via VTILVOT in TOE and by thermodilution using PAC. Methods In 167 patients undergoing coronary artery bypass graft surgery (CABG), TTE cine loops of apical 4- and 2-chamber views were recorded after anaesthesia induction under steady-state conditions. Subsequently, TOE was performed following a standardized protocol, and CO was determined by thermodilution. EF and CO were assessed by TTE AutoEF as well as TOE, using the modified Simpson’s method, and Doppler measurements via velocity time integral in the LV outflow tract (VTILVOT). We determined Pearson’s correlation coefficients r and carried out Bland–Altman analyses. The primary endpoints were differences in EF and CO. The secondary endpoints were differences in left ventricular volumes at end diastole (LVEDV) and end systole (LVESV). Results AutoEF and the modified Simpson’s method in TOE showed moderate EF correlation (r = 0.38, p < 0.01) with a bias of -12.6% (95% limits of agreement (95%LOA): -36.6 – 11.3%). AutoEF CO correlated poorly both with VTILVOT in TOE (r = 0.19, p < 0.01) and thermodilution (r = 0.28, p < 0.01). The CO bias between AutoEF and VTILVOT was 1.33 l min−1 (95%LOA: -1.72 – 4.38 l min−1) and 1.39 l min−1 (95%LOA -1.34 – 4.12 l min−1) between AutoEF and thermodilution, respectively. AutoEF yielded both significantly lower EF (EFAutoEF: 42.0% (IQR 29.0 — 55.0%) vs. EFTOE Simpson: 55.2% (IQR 40.1 — 70.3%), p < 0.01) and CO values than the reference methods (COAutoEF biplane: 2.30 l min−1 (IQR 1.30 - 3.30 l min−1) vs. COVTI LVOT: 3.64 l min−1 (IQR 2.05 - 5.23 l min−1) and COPAC: 3.90 l min−1 (IQR 2.30 - 5.50 l min−1), p < 0.01)). Conclusions AutoEF correlated moderately with TOE EF determined by the modified Simpson’s method but poorly both with VTILVOT and thermodilution CO. A systematic bias was detected overestimating LV volumes and underestimating both EF and CO compared to the reference methods. Trial registration German Register for Clinical Trials (DRKS-ID DRKS00010666, date of registration: 08/07/2016).