The aim of this study was to investigate the effectiveness of guidance assistance during transbronchial biopsy (TBB) to achieve an appropriate pathway to small and peripheral pulmonary lesions (PPLs) using a combination of fluoroscopy and specialized ray-summation (Ray-sumTBB) images, which were processed from preprocedural lung computed tomography (CT) images. To improve the visibility of the correct pathway to the PPLs, three-dimensional spatial resolution enhancement and CT number conversion processes were applied to the original CT images. The Ray-sumTBB images reconstructed from the processed CT images were used as additional guides. We compared the rates of successful tumor localization and biopsy (arrival rate) between the trial (with Ray-sumTBB) and control (without Ray-sumTBB) groups. The fluoroscopy and examination times were also compared. The arrival rate of the trial group (73.1%) was significantly better than that of the control group (42.3%) (p = 0.048). The fluoroscopy and examination times did not differ significantly between the trial and control groups. No complications were identified in the trial group. Our results suggest that Ray-sumTBB improves the diagnostic accuracy of TBB.