This dataset comprises raw data from research investigating the application of nisin and lactoferrin as potential novel antimicrobial agents in dentistry. The data are organized in Microsoft Excel spreadsheets and include results from the following assays: (1) minimum inhibitory concentration (MIC); (2) minimum bactericidal concentration (MBC); (3) checkerboard assays for the evaluation of antimicrobial interactions; (4) reduction of mature biofilms; (5) cytotoxicity assays in human gingival fibroblasts; and (6) physicochemical and microbiological evaluation of the developed topical formulation. Additional metadata are provided within the files to support data interpretation, including detailed information on the concentrations of the tested substances, experimental groups, and bacterial and cell culture conditions (gingival fibroblasts). These data enable qualitative and quantitative analyses of the antimicrobial and cytotoxic effects of nisin and lactoferrin, in comparison with chlorhexidine digluconate.