Aims: Evaluation of fermentation process parameter interactions for the production of L-asparaginase by isolated Staphylococcus sp. - 6A. Methods and Results: Fractional factorial design of experimentation (L18 orthogonal array of Taguchi methodology) was adopted to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. The experimental results and software predicted enzyme production values were comparable. Conclusions: Incubation temperature, inoculum level and medium pH, among all fermentation factors, were major influential parameters at their individual level, and contributed to more than 60% of total L-asparaginase production. Interaction data of selected fermentation parameters could be classified as least and most significant at individual and interactive levels. Aeration and agitation were most significant at interactive level, but least significant at individual level, and showed maximum severity index and vice versa at enzyme production. Significance and Impact of the Study: All selected factors showed impact on L-asparaginase enzyme production by this isolated microbial strain either at the individual or interactive level. Incubation temperature, inoculum concentration, pH of the medium and nutritional source (glucose and ammonium chloride) had impact at individual level, while aeration, agitation and incubation time showed influence at interactive level. Significant improvement (ninefold increase) in enzyme production by this microbial isolate was noted under optimized environment.