In this study, the performance of a new iterative reconstruction algorithm, the pre-clinical AcurosXB iCBCT algorithm, has been characterized on Varian Halcyon linear accelerators with respect to the potential of radiotherapy dose calculations on CBCT images. The study utilized various phantom setups to verify the accuracy of the pre-clinical algorithm under different scatter conditions and compared dose calculations performed on CBCT images reconstructed with the pre-clinical algorithm to those performed on typical planning CT images. The results indicated that despite showing improvements compared to the existing iCBCT protocol, certain restrictions should be introduced when the pre-clinical AcurosXB iCBCT algorithm was used for dose calculations. Changes in the scatter condition exhibited a larger effect on CBCTs than on planning CTs. Therefore, users should be careful in offsetting the patient and positioning the patient’s arms if the resultant images will be used for dose calculations. In addition, protocols with different kV settings should be approached with caution, where 100 kV protocols should only be used to scan the head and neck area, while the rest of the body should be scanned with the 125 kV and 140 kV protocols. When the patient is set up properly and the appropriate energy is selected for the anatomical area, the uncertainty of using the novel AcurosXB iCBCT algorithm for treatment planning dose calculation is within ± 2.0%.