Detecting microcalcifications (MCs) in real time is important in the guidance of many breast biopsies. Due to its capability in visualizing biopsy needles without radiation hazards, ultrasound imaging is preferred over X-ray mammography, but it suffers from low sensitivity in detecting MCs. Here, we present a new nonionizing method based on real-time multifocus twinkling artifact (MF-TA) imaging for reliably detecting MCs. Our approach exploits time-varying TAs arising from acoustic random scattering on MCs with rough or irregular surfaces. To obtain the increased intensity of the TAs from MCs, in MF-TA, acoustic transmit parameters, such as the transmit frequency, the number of focuses and f-number, were optimized by investigating acoustical characteristics of MCs. A real-time MF-TA imaging sequence was developed and implemented on a programmable ultrasound research system, and it was controlled with a graphical user interface during real-time scanning. From an in-house 3D phantom and ex vivo breast specimen studies, the MF-TA method showed outstanding visibility and high-sensitivity detection for MCs regardless of their distribution or the background tissue. These results demonstrated that this nonionizing, noninvasive imaging technique has the potential to be one of effective image-guidance methods for breast biopsy procedures.