Introduction Del Nido cardioplegia (DN) is gaining acceptance in adult cardiac surgery but there is paucity of experimental data regarding its efficacy. We set out to assess the safety and efficacy of single-dose DN with and without topical cooling (TC) versus multi-dose blood cardioplegia (BC). Methods Thirty-two healthy adult sheep had pressure-volume (PV) catheters placed in the left (LV) and right (RV) ventricle. Animals were assigned to receive cold (4°C) antegrade solution for a 60-min arrest using: (1) multi-dose (every 20 min) BC with TC ( n = 11), (2) single-dose DN with TC (DN-C, n = 10), or (3) single-dose DN without TC (DN-H, n = 11). LV and RV PV-derived indexes, epicardial echocardiographic strains, and blood samples were acquired before CPB and at 1, 2, and 3 h of reperfusion. Dobutamine bolus (2.5 μg) was given after 3 h to test for myocardial reserve. Results Time to rhythm restoration was shortest (54 ± 29 s, 118 ± 167 s, and 172 ± 170 s for DN-H, DN-C, and BC, respectively; p = 0.024) and number of shocks lowest (1.7 ± 1.8, 3.6 ± 2.8, and 5.6 ± 4.6 for DN-H, DN-C, and BC, respectively; p = 0.020) in DN-H group. Hemodynamic, load-independent myocardial function, echocardiographic, and metabolic data revealed only slight differences between groups. Troponin I levels did not differ between groups. With dobutamine, preload-recruitable stroke work of both LV (136 ± 50%, 131 ± 31%, 142 ± 58% for BC, DN-C and DN-H, respectively; p = 0.993) and RV (161 ± 67%, 185 ± 45%, 166 ± 75% for BC, DN-C and DN-H respectively; p = 0.580) increased similarly. Conclusions Single-dose DN cardioplegia with or without topical cooling offered comparable biventricular myocardial protection to multi-dose BC for a 60-min arrest in sheep.