Bacterial resistance to antibiotics is considered the most important health problem in developed and developing countries. Studies on new pharmaceutical strategies, such as probiotics and their derivatives, have increased. The purpose of this study is to isolate and purify bacteriocin and biosurfactant from Lactobacillus rhamnosus and investigate its antibacterial and anti-biofilm activity. Bacteriocin and biosurfactant produced by Lactobacillus rhamnosus standard strain were isolated and purified. Biosurfactant screening tests, including oil expansion test and drop spreading test, were conducted. Bacteriocin was separated using two different methods, including separation with the help of 2-propanol and separation with the help of ammonium sulfate. The purification of bacteriocin was realized by dialysis, and the effect of biosurfactant and bacteriocin on Staphylococcus aureus and Escherichia coli was realized using the disc diffusion method. The antibacterial properties of these substances were determined by diffusion method from its anti-biofilm disk with the help of the ELISA reader. Statistical Program for Social Sciences (SPSS) software was utilized for data analysis. The diameter of the non-growth halo of E.coli in contact with the disc impregnated with bacteriocin in concentrations of 250, 500, and 1000mg/ml was 21 +/- 1, 21.33 +/- 2.3 and 34.66 +/- 7.02 mm, respectively, and in Staphylococcus aureus was evaluated as 18.33 +/- 3.78, 24.33 +/- 4.72 and 43.66 +/- 4.72 mm, respectively. The order was evaluated as 18.33 +/- 2.88, 25 +/- 5, 43.33 +/- 5.77 mm. The biofilm inhibition percentage was measured for Escherichia coli 0.36, 2.15, and 71.75%, respectively, and for Staphylococcus aureus 1.07%, 5.96%, and 53.3%, respectively, at the mentioned concentrations. The results showed that bacteriocin and biosurfactant isolated from Lactobacillus rhamnosus have high antibacterial and anti-biofilm properties.