The purpose of this clinical study was to assess, in a limited patient population, the potential for a novel advanced wound care treatment based on low-frequency (20 kHz) low-intensity (spatial peak temporal peak intensity <100 mW/cm2; i.e., pressure amplitude of 55 kPa) ultrasound (LFLI-US), to affect wound closure rate in human diabetic foot ulcers (DFUs) and to effect changes in the relative expression of pro-inflammatory and anti-inflammatory genes. The ratio of expression of these genes, termed the M1/M2 score because it was inspired by the transition of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) phenotypes as wound healing progresses, was previously presented as a potential healing indicator for DFUs treated with the standard of care. We previously found that non-cavitational, non-thermal LFLI-US delivered with a pulse repetition frequency of 25 Hz was effective at improving wound healing in a pilot study of 20 patients with chronic venous ulcers. In this study, we assessed the potential for weekly LFLI-US exposures to affect wound healing in patients with diabetic ulcers, and we analyzed temporal changes in the M1/M2 score in debrided diabetic wound tissue. Although this was a limited patient population of only 8 patients, wounds treated with LFLI-US exhibited a significantly faster reduction in wound size compared with sham-treated patients (p < 0.001). In addition, the value of the M1/M2 score decreased for all healing diabetic ulcers and increased for all non-healing diabetic ulcers, suggesting that the M1/M2 score could be useful as an indicator of treatment efficacy for advanced DFU treatments. Such an indicator would facilitate clinical decision making, ensuring optimal wound management and thus contributing to reduction of health care expenses. Moreover, the results presented may contribute to an understanding of the mechanisms underlying ultrasonically assisted chronic wound healing. Knowledge of these mechanisms could lead to personalized or patient-tailored treatment.