This paper presents the use of an effluent derived from two-stage anaerobic digestion of two-phase olive mill solid waste (OMSW) as a substrate for the production of Chlorella zofingiensis in batch mode. Chlorella zofingiensis when grown autotrophycally can accumulate significant quantities of valuable carotenoids which are used as an additive in fish and poultry farming, as colorants in foods and in health care products. It was found that two-phase OMSW previously treated by two-stage anaerobic digestion and further sterilized may be used as a culture medium for the microalgae Chlorella zofingiensis. Typical growth curves were obtained using both the above-mentioned anaerobic effluent and a synthetic medium. Total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (SCOD) removals of 37% and 45% respectively were achieved in batch experiments after 11 days’ operation time. The specific growth rate was lower when the treated effluent was used as the feed substrate (0.02 h-1) in comparison to the synthetic medium (0.03 h-1). The specific growth rates of the exponential phases were determined by using a first-order kinetic model applied to chlorophyll a (Ca) and total chlorophyll (TC) concentrations, as indirect measurements of the microalgae concentration. It was concluded that the effluent from two-stage anaerobic digestion of two-phase OMSW constituted an appropriate culture medium for the growth of Chlorella zofingiensis, providing a simple technology feasible for producing a very useful product for animal feeding.