Background: Mammography is essential for early diagnosis of breast cancer, which is the most common type of cancer in females that is associated with a high mortality rate. We investigated whether evaluation of the grayscale inverted images of mammograms would aid in increasing the diagnostic sensitivity of the mammographic imaging technique. Methods: Our study included 636 mammograms of 159 women who had undergone digital mammography. Standard, grayscale inverted, and standard plus grayscale inverted images were sequentially examined three times, at 15-day intervals, for the presence or assessment of pathological changes in the skin, calcification, asymmetric density, mass lesions, structural distortions, and intramammary and axillary lymph nodes. To determine whether grayscale inverted image assessment improved detection rates, the results of the three assessment modes were compared using Cochran's Q test and the McNemar test (p < 0.05 was considered statistically significant). Results: The average age of 159 patients was 50.4 years (range, 35-80 years). There were significant differences among the three assessment modes with respect to calcification and intramammary lymph nodes (p < 0.05); however, no significant differences were observed for the detection of other parameters. Conclusions: Assessment of grayscale inverted images in addition to standard images facilitates the detection of microcalcification.