ABSTRACT Clostridial collagenase is recognized as one of the important proteolytic enzymes used in treatment of varieties of fibro-proliferative disorders. The present work aimed to optimise the biomass concentration and collagenase enzyme activity of novel bacilli Clostridium novyi-NT. The response surface methodology tool was used to identify the optimal fermentation parameters. Central composite design (CCD) was applied with respect to three influencing factors - pH, proteose peptone and trypticase soya broth. These factors showed significant effect on the overall biomass concentration and collagenase enzyme activity (p<0.05). The maximum biomass concentration in terms of absorbance and collagenase activity (of crude enzyme) was achieved and recorded as 0.8309±0.0012 and 298.88±1.36 units/mg respectively after 22 hours of fermentation period while optimisation of media factors with help of response surface quadratic model. This is the first study to report maximum biomass concentration and collagenase activity by Clostridium novyi-NT till date by combining statistical designs.