Tendon augmentation is increasingly clinically relevant due to rising amount of tendon ruptures because of the aging and more demanding population. Therefore, newly developed scaffolds based on bovine epoxide stabilized collagen maintaining the native fibril-like collagen structure were characterized and compared to two commercially available porcine collagen scaffolds. For biomechanical testing (ultimate load, ultimate stress, stiffness, and elastic modulus), bovine collagen scaffolds were hydrated and compared to reference products. Cell viability and proliferation were assessed by seeding human primary fibroblasts on each collagen-based scaffold and cultured over various time periods (3 d, 7 d, and 14 d). Live/dead staining was performed and metabolic cell activity (WST-1 assay) was measured. Biochemical degradability was investigated by enzymatic digestion. The bovine collagen scaffold showed significantly enhanced biomechanical properties. These persisted over different rehydration times. Cell biological tests revealed that the bovine collagen scaffolds support reproducible cell colonization and a significant increase in the number of viable cells during cultivation. The results are comparable with the viability and proliferation rate of cells grown on porcine reference materials. With regard to biochemical degradability, all tested materials showed comparable resistance to enzymatic degradation in vitro. Due to imitating the natural tendon structure the new scaffold material is supposed to provide beneficial effects in future clinical application.