Purpose: Hyperthermic ablation is a minimally invasive mode of tumour therapy which serves as a viable alternative to surgical intervention. However, one of the major drawbacks, besides the heat sink effect and the risk of damaging adjacent organs, is limited ablation size. The use of a cooling fluid during ablation has been shown to increase the ablation volume and decrease the carbonisation rate. The aim of this study was to investigate whether the composition of the cooling fluid has an effect on ablation size and carbonisation rate during hepatic laser ablation in an ex vivo bovine setting. Method: In this study bovine hepatic tissue was ablated in an ex vivo setting using an internally cooled laser applicator. A total of 45 tissue samples were assigned to three groups: 0.9% saline infusion (n = 15), distilled water infusion (n = 15) and a 50%/50% mixture of 0.9% saline and distilled water (n = 15). Ablation was conducted using a 1064 nm Nd:YAG laser at a wattage of 25 W and time interval of 10 min. The ablation volume and carbonisation rate were then measured and recorded through postprocedural MRI. One-way ANOVA and post-hoc testing were performed to assess the effect of the cooling fluid composition on the ablation volumes. Results: We found that using a mixture of saline and distilled water as a cooling fluid during hyperthermic ablation resulted in a larger ablation volume (mean ± SD: 22.64 ± 0.99 cm3) when compared to saline infusion (21.08 ± 1.11 cm3) or distilled water infusion (20.92 ± 0.92 cm3). This difference was highly significant (p < 0.001). There was no significant difference in ablation size between the saline group and the distilled water group. The highest carbonisation rate occurred in the saline group (12/15), followed by the mixed infusion group (3/15) and the distilled water group (1/15). Conclusions: The results of this study suggest that cooling fluid composition during hepatic laser ablation affects ablation volume in an ex vivo bovine setting. There was no statistically significant difference when comparing ablation volumes during saline infusion and distilled water infusion, but the carbonisation rate was significantly higher when using saline. The combination of saline and distilled water in a 50%/50% mixture as cooling fluid appears to be an auspicious alternative, as ablation volumes created with it are larger when compared to saline and distilled water alone, while carbonisation rate remains low. This might improve patient outcome as well as patient eligibility for hyperthermic ablation.