Re-excision due to inability to visualize positive margins following breast-conserving surgery is a significant clinical challenge. 5-aminolevulinic acid (5-ALA), a non-fluorescent prodrug, leads to intracellular accumulation of fluorescent porphyrins in tumor cells. This single-centre Phase II randomized controlled trial evaluated the clinical safety, feasibility and diagnostic performance of a new handheld fluorescence imaging device (PRODIGI) combined with 5-ALA hydrochloride (HCl) for intraoperative visualization of invasive breast carcinomas. Fifty-four patients were enrolled in the study and randomized to receive no 5-ALA HCl or orally administered 5-ALA HCl (15 mg/kg or 30 mg/kg BW). Forty-five patients (n = 15/group) were included in the analysis. Fluorescence imaging of the excised surgical specimen was performed and biopsies were collected from within and outside the clinically-demarcated tumor border for blinded histopathological analysis. In the absence of 5-ALA HCl, tissue autofluorescence imaging lacked tumor-specific fluorescent contrast. Administration of 5-ALA HCl resulted in tumors that fluoresced bright red with improved visualization of tumor contrasted against normal tissue autofluorescence. In the 15 mg/kg 5-ALA dose group the positive predictive value (PPV) for detecting tumor inside and outside the grossly demarcated tumor border was 100.0% and 55.6%, respectively and 100.0% and 50.0% respectively in the 30 mg/kg dose group. No drug or device-related adverse events were observed and technical feasibility and clinical integration of this intraoperative tumor visualization approach were confirmed. This is the first known clinical report of visualization of 5-ALA HCl-induced fluorescence in invasive lobular and ductal breast carcinoma using a real-time handheld intraoperative fluorescence imaging device.