The relationship between bacteria and venous ulceration pain is well-established and primarily attributable to inflammatory pathways. Fluorescence imaging detects clinically significant bacterial loads and biofilm in real time at the bedside, informing its elimination in an objective manner. We sought to explore the regional co-localization of bacterial fluorescence signals and patient-reported venous ulceration pain, and if objectively targeted bacterial removal can reduce wound-associated pain.We evaluated 46 adults with venous ulceration of the lower extremity self-reporting a wound-associated pain score of ≥4 on a scale of 1 to 10. Before any treatments were performed (eg, debridement), patients rated their pain during the study visit, and fluorescence images were captured. Regions of pain and positive fluorescence signals were sketched onto a printed wound image. Fluorescence imaging was repeated post procedurally, and patients rerated their pain either at the end of the study visit or over the phone the following day. Semiquantitative analysis involved visual estimation of the percentage overlap between regions of fluorescence and pain in the wound bed. Wilcoxon matched pairs signed rank tests and Mann-Whitney t tests assessed changes in pain scores post procedurally.Fluorescence from elevated bacterial loads and biofilm was present in every venous ulcer assessed, usually covering ≤50% of the wound bed and commonly colonizing the wound edges. Regions of pain were more extensive than regions of fluorescence within the wound bed, and some degree of overlap was identified in 40 of 46 patients (87%). This overlap was often substantial (29 patients with >25% overlap and 16 with >50% overlap). Overall mean pain scores were 8.17 before the procedure and 6.87 after the procedure, corresponding with a 1.30-point reduction that was highly statistically significant (P < .0001). Pain score reduction was higher when patients rerated their pain 1 day after debridement (3.40-point reduction; P = .004).We observed that fluorescence signals from clinically significant bacterial colonization and biofilms were commonly present in painful venous lower extremity ulcerations. Regions of patient-reported pain and positive fluorescence frequently overlapped, suggesting a relationship between the two. Wound-associated pain scores were significantly and immediately reduced after objectively targeted bacterial removal via real-time fluorescence imaging, with an even greater reduction observed by the next day. Understanding the association between chronic bacterial presence and pain in venous ulcers can inform treatment and management strategies, potentially enhancing patient quality of life and satisfaction, promoting healing, and reducing complications.