Background Transpedicular biopsy of spinal lesions is imperative for the generation of a definite diagnosis. Thus far, literature comparing the accuracy and adequacy between fluoroscopy-guided and computed tomography (CT)-guided transpedicular biopsy of spinal lesions is scarce. We aim to compare the accuracy and adequacy of samples collected with the two techniques at the largest tertiary hospital in Malaysia. Materials and methods A total of 60 patients (37 patients with spinal infection and 23 patients with spinal tumour) underwent percutaneous transpedicular biopsy of spinal lesions from January 2013 to December 2017 at a tertiary centre. Demographic data, biopsy method (fluoroscopy-guided and CT-guided), diagnosis, adequacy, and accuracy of samples obtained were assessed. Results Among the 60 samples obtained, only two samples (3.3%) were deemed inadequate. There were 10 biopsy samples (16.7%) that were inaccurate. There was no statistical difference between fluoroscopy-guided and CT-guided transpedicular biopsy in terms of accuracy (p = 0.731) and adequacy (p = 0.492). Conclusions Fluoroscopy-guided and CT-guided transpedicular biopsy of spinal lesions offer similar accuracy and adequacy. Fluoroscopy-guided biopsy of the spinal lesion will be an option for spine surgeons when CT-guided biopsy is not available.