Purpose To compare the diagnostic performance of cone-beam CT (CBCT)-guided and CT fluoroscopy (fluoro-CT)-guided technique for transthoracic needle biopsy (TNB) of lung nodules. Methods The hospital records of 319 consecutive patients undergoing 324 TNBs of lung nodules in a single radiology unit in 2009–2013 were retrospectively evaluated. The newly introduced CBCT technology was used to biopsy 123 nodules; 201 nodules were biopsied by conventional fluoro-CT-guided technique. We assessed the performance of the two biopsy systems for diagnosis of malignancy and the radiation exposure. Results Nodules biopsied by CBCT-guided and by fluoro-CT-guided technique had similar characteristics: size, 20 ± 6.5 mm (mean ± standard deviation) vs. 20 ± 6.8 mm ( p  = 0.845); depth from pleura, 15 ± 15 mm vs. 15 ± 16 mm ( p  = 0.595); malignant, 60 % vs. 66 % ( p  = 0.378). After a learning period, the newly introduced CBCT-guided biopsy system and the conventional fluoro-CT-guided system showed similar sensitivity (95 % and 92 %), specificity (100 % and 100 %), accuracy for diagnosis of malignancy (96 % and 94 %), and delivered non-significantly different median effective doses 11.1 mSv (95 % CI 8.9–16.0) vs. 14.5 mSv (95 % CI 9.5–18.1); p  = 0.330. Conclusion The CBCT-guided and fluoro-CT-guided systems for lung nodule biopsy are similar in terms of diagnostic performance and effective dose, and may be alternatively used to optimize the available technological resources. Key Points • CBCT-guided and fluoro-CT-guided lung nodule biopsy provided high and similar diagnostic accuracy. • Effective dose from CBCT-guided and fluoro-CT-guided lung nodule biopsy was similar. • To optimize resources, CBCT-guided lung nodule biopsy may be an alternative to fluoro-CT-guided.