Despite advancements in the surgical techniques of rotator cuff repair (RCR), there remains a high retear rate. Biological augmentation of repairs with overlaying grafts and scaffolds may enhance healing and strengthen the repair construct. This study aimed to investigate the efficacy and safety of scaffold-based (nonstructural) and overlay graft-based (structural) biological augmentation in RCR (excluding superior capsule reconstruction and bridging techniques) in both preclinical and clinical studies./r/nThis systematic review was performed in adherence to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, as well as guidelines outlined by The Cochrane Collaboration. A search of the PubMed, Embase, and Cochrane Library databases from 2010 until 2022 was conducted to identify studies reporting the clinical, functional, and/or patient-reported outcomes of ≥1 biological augmentation method in either animal models or humans. The methodologic quality of included primary studies was appraised using the Checklist to Evaluate a Report of a Non-pharmacological Trial (CLEAR-NPT) for randomized controlled trials and using the Methodological Index for Non-randomized Studies (MINORS) for nonrandomized studies./r/nA total of 62 studies (Level I-IV evidence) were included, comprising 47 studies reporting outcomes in animal models and 15 clinical studies. Of the 47 animal-model studies, 41 (87.2%) demonstrated biomechanical and histologic enhancement with improved RCR load to failure, stiffness, and strength. Of the 15 clinical studies, 10 (66.7%) illustrated improvement in postoperative clinical, functional, and patient-reported outcomes (eg, retear rate, radiographic thickness and footprint, and patient functional scores). No study reported a significant detriment to repair with augmentation, and all studies endorsed low complication rates. A meta-analysis of pooled retear rates demonstrated significantly lower odds of retear after treatment with biological augmentation of RCR compared with treatment with non-augmented RCR (odds ratio, 0.28; P < .00001), with low heterogeneity (I = 0.11)./r/nGraft and scaffold augmentations have shown favorable results in both preclinical and clinical studies. Of the investigated clinical grafts and scaffolds, acellular human dermal allograft and bovine collagen demonstrate the most promising preliminary evidence in the graft and scaffold categories, respectively. With a low risk of bias, meta-analysis revealed that biological augmentation significantly lowered the odds of retear. Although further investigation is warranted, these findings suggest graft and scaffold biological augmentation of RCR to be safe.