Cotton stalk lignin extractions were performed via alkaline methods at different conditions. Crude and post treated cotton stalk lignins, olive mill wastewater and garlic stalk juice were examined in terms of antimicrobial activity. Antimicrobial lignin was isolated depending on alkaline extraction conditions. Lignin extracted at 60&deg / C exhibited significant antimicrobial effect towards both Escherichia coli and Bacillus pumilus. However different post treatments such as ultrasonication and TiO2-assisted photocatalytic oxidation did not result in antimicrobial compounds. Olive mill wastewater and garlic stalk juice exerted substantial antimicrobial effects towards tested microorganisms. Xylan-based biodegradable films containing lignin, garlic stalk juice, tannic acid and olive mill wastewater were characterized against both B. pumilus and E. coli by means of their antimicrobial activities. E. coli exhibited lesser sensitivity to all tested antimicrobial xylan films except tannic acid-integrated xylan film than B. pumilus. Antimicrobial lignin integrated-xylan film exhibited stronger effect towards tested microorganisms than tannic acid-integrated film. In the case of both antimicrobial lignin and tannic acid integrated xylan films, 4% was found to be the maximum antimicrobial compound percentage in film forming solutions to observe continuous film formation. Lignin samples with/without antimicrobial activity were characterized by means of their chemical structure via FTIR and LC-MS. FTIR results revealed that cotton stalk lignins were significantly broken down via alkaline treatment and this breakdown resulted in the formation of new fractions and also ester &amp / ether bonds between antimicrobial hydroxycinnamic acids and lignin were cleaved during the alkaline treatments of cotton stalk lignins. By FTIR results, C=C bonds were found to be characteristic for antimicrobial lignin sample and it was suggested that these bonds might be the reason of the antimicrobial activity. By LC-MS qualitative mass analysis, antibacterial lignin fractions were found to be quite different from non-antibacterial lignin fractions. LC-MS results indicated that the antimicrobial lignin fractions might be lignin-derived oligomers and/or might be flavonoids. Cotton stalk lignin fractions demonstrated different antimicrobial activities depending on the method of isolation and chemical treatment.