We evaluated the antifungal and anti-biofilm activity, mechanism of action and cytotoxicity of chloramine T trihydrate (CAT) against spp. The Minimum Inhibitory and Fungicidal Concentrations (MIC/MFC) of CAT were determined. Changes in CAT-treated growth kinetics and micromorphology were evaluated, as well as the mechanism of action, and its effects on biofilm. Cytotoxicity was assessed by the hemolysis method. The data were analyzed by inferential statistics ( ≤ 0.05). CAT showed antifungal activity against all strains, with MIC values ranging between 1.38 and 5.54 mmol/L (MIC: 2.77 mmol/L). CAT demonstrated an immediate and sustained action on growth kinetics, particularly at 2 × MIC. This compound likely acts on the cell wall and membrane permeability simultaneously and was found to cause changes in micromorphology. Tha antibiofilm activity of CAT was similar to that of sodium hypochlorite ( > 0.05) against mature biofilms. CAT was more effective than NaOCl in reducing mature biofilm upon 1-min exposure at 2 × MIC (24 h) and 4 × MIC (48 h) ( < 0.05). Toxicological analysis revealed that CAT had hemolytic activity between 61 and 67.7% as compared to 100% by NaOCl. CAT has antifungal and anti-biofilm properties, probably acting on both cell wall and membrane permeability, and showed low toxicity in vitro.