Bacillus sphaericus has been widely used in mosquito control programs, but the production of this bacterium is a little tricky as it does not utilize carbohydrates and requires proteinaceous substrates, which are expensive. In this study, we developed a cost-effective medium that resulted in a lower cost and shorter fermentation time. The locally available raw material, egg yolk was used and the level of sporulation, toxicity and biomass were compared with the conventional medium. Use of the egg yolk culture medium significantly shortened fermentation time to 15 h and yielded high activity, equivalent to that of conventional medium against 3rd instar Culex quinquefasciatus. Conventional NYSM medium required 21 h to attain the maximum activity and biomass. Hence, the egg yolk-based culture medium appears to be suitable and economical for the small-scale production of B. sphaericus.