Aim:The aim of this study is to evaluate efficacy of various combinations of Chlorhexidine And Povidone-iodine using anti-microbial ,anti-inflammatory and cytotoxicity tests. Material and Methods:Commercially available pure compounds of 20% chlorhexidine gluconate and 99% Povidone Iodine were ordered from Sigma Aldrich.Out of which 0.2% concentration of both compounds were prepared by mixing 9.8ml of distilled water and 0.2ml of Chlorhexidine Gluconate and 10 ml of distilled water with 0.2gm of Povidone Iodine.Prepared samples went under anti-microbial testing in 3 wound pathogens and 3 oral pathogens, Anti Inflammatory testing was carried out in Bovine serum albumin each group had 5 samples or 10,20,30,40,50 microlitre, Cytotoxicity test was carried out in brine shrimps each group had 5 samples 5,10,20,40,80 microlitre ELISA plates were loaded with 10 shrimps each. Results:There was a significant difference (P<0.05) of zone of inhibition , anti-inflammatory activity and cytotoxicity among all the groups against all microbes selected for the study where the combination of PI and CHX in different concentrations was showing better activity than PI or CHX alone and the difference was statistically significant (p<0.05, derived from post hoc TuKey). Conclusion:Combination of chlorhexidine and povidone iodine has proven to be superior to using these antibiotics alone but as this is an in vitro study ,similar results have to be verified with clinical trials to prove efficacy of the combination over these antibiotics