The aim of our study is to evaluate preserving gracilis tendon in anterior cruciate ligament reconstruction (ACLR) surgery and its effect to the flexion of the knee joint and tibial internal rotation strength and the stability of the knee. Patients who underwent primary single-bundle arthroscopic ACLR using all-inside technique and using hamstring tendon autograft were evaluated retrospectively. Patients were divided into two groups as gracilis preserved (St) and gracilis harvested (StG) groups. The International Knee Documentation Committee (IKDC) score, Lysholm, Knee Injury and Osteoarthritis Outcome Score—Knee-related quality of life (KOOS-QOL) score, ACL-Return to Sport after Injury scale score were used to evaluate as postoperative functional scores at last follow-up. Anterior tibial translation was evaluated using the KT-1000 device. Knee joint flexion, extension, and internal rotation strength were evaluated using isokinetic dynamometer. Dynamic balance performances were measured using the Biodex Balance System. There were 24 patients in the St group and 23 patients in the StG group. Demographic data and clinical results showed no significant difference. Anteroposterior movement of the tibia was found to be significantly higher in the StG group than in the St group in measurements at 89 and 134 N, respectively (p = 0.01 and <0.001). No statistically significant difference was found between both standard and deep flexor and extensor and internal rotator strength. No statistically significant difference was found in the amount of total, anteroposterior, and mediolateral balance deficit between the two groups. Additional gracilis harvesting does not have a negative effect on both standard and deep knee flexion, and tibial internal rotation strength compared with the St group. Although semitendinosus and StG group showed significantly more anterior tibial translation, there was no significant difference in clinical and dynamic stability measurements.