Approximately 20–30% of breast-conserving surgery (BCS) patients require follow-up re-excision surgery due to inadequate cancer resection. To aid cancer detection and resection, we present a handheld optical coherence tomography (OCT) probe and attenuation imaging method to enable in vivo imaging of the surgical cavity during BCS. The attenuation imaging method incorporates three-dimensional correction of system factors in the probe, namely, confocal function and sensitivity roll-off function, demonstrating accurate and repeatable attenuation imaging of phantoms, validated using the Mie scattering model. To assess the imaging performance of the probe prior to in vivo imaging, ex vivo attenuation imaging of breast specimens from eight mastectomy patients was performed and the results compared to those acquired using a benchtop imaging system, showing consistent tissue contrast in attenuation images. In vivo imaging of breast tissue in the surgical cavity of seven patients was subsequently performed during BCS. The results demonstrate the potential of attenuation imaging to delineate cancer in vivo in the surgical cavity. Attenuation imaging provides an additional tissue contrast mechanism to complement OCT imaging, potentially contributing to more complete identification and resection of cancer during BCS.