Although the rate of infection after the reconstruction of a ruptured anterior cruciate ligament (ACL) is low, prophylactic incubation of the graft with vancomycin (Vanco-wrap or vancomycin soaking) is routinely performed. A cytotoxic effect of vancomycin is reported for several cell types, and the prophylactic treatment might prevent infection but harm the tissue and cells. Aim: A comprehensive study was performed to investigate the effect of vancomycin on tendon tissue and isolated tenocytes using cell viability, molecular and mechanical analysis. Material and methods: Rat tendons or isolated tenocytes were incubated in increasing concentrations of vancomycin (0–10 mg/mL) for different times, and cell viability, gene expression, histology and Young’s modulus were analyzed. Results: The clinically used concentration of vancomycin (5 mg/mL for 20 min) had no negative effect on cell viability in the tendons or the isolated tenocytes, while incubation with the toxic control significantly reduced cell viability. Increasing the concentration and prolonging the incubation time had no negative effect on the cells. The expression ofCol1a1,Col3a1and the tenocyte markersmohawk,scleraxisandtenomodulinwas not affected by the various vancomycin concentrations. The structural integrity as measured through histological and mechanical testing was not compromised. Conclusion: The results proved the safe application of the Vanco-wrap on tendon tissue. Level of evidence: IV.