In vivo estimation of attenuation coefficients is useful because of its potential for tissue characterization and relevance in accurate backscatter coefficient estimation. Recently, the regularized spectral log difference (RSLD) technique was proposed to extend the trade-off between precision and spatial resolution in attenuation coefficient slope (ACS) estimation. The aim of this pilot study is to validate the feasibility of in vivo estimation of ACSs from thyroid nodules using the RSLD technique. In vivo data from thyroid nodules was acquired in an oncology clinic right before fine needle aspiration biopsy (FNAB) procedure. Six nodules were analyzed in this study having FNAB results reported as either Bethesda II (benign) or VI (cancer). The nodules had an average diameter of 1.56 cm and their biopsy results were adenomatoid nodules (three cases), Hashimoto's thyroiditis (two cases), and papillary carcinoma (one case). In addition, healthy thyroid regions in three additional patients were also evaluated. The ACS estimation was performed with 16 wavelength axially by 16 wavelengths laterally data blocks. The average ACS values in healthy thyroid tissues and Hashimoto's thyroiditis nodules were found to be 1.62 ± 0.27 and 0.97 ± 0.08 dB.cm-1.MHz-1, which are consistent with previous reports in this subject in the literature. The ACS values for the adenomatoid nodules and the papillary carcinoma were 0.94 ± 0.07 and 0.87 dB.cm-1.MHz-1, respectively. All nodules had lower ACS values than the ones in healthy thyroid tissues. These results suggest that the RSLD method has potential for producing accurate and precise estimates of ACS values in vivo.