Microbial alkalinity production was evaluated as a method to prevent reacidification of neutralized mining lakes by acidic ground and seepage water. We used 60 L mesocosms to represent the sediment and water column of a shallow acidic mine lake. To enhance alkalinity production, acidic and neutralized lake waters were treated with either phosphorus (controlled eutrophication) or organic matter (controlled saprobization). Controlled eutrophication could not produce enough autochthonous biomass as substrate for microbial alkalinity production to change the acidity of the water. Chemical pre-neutralization of the acidic water caused the inorganic carbon concentration to increase, but at the same time, hindered algae growth by reducing the availability of phosphate by sorption to the freshly precipitated iron hydroxide. This effect was so strong that even high phosphorus additions could not increase the algae biomass production. In contrast to controlled eutrophication, controlled saprobization produced significant alkalinity. Despite inhibition of the most important alkalinity producing process, namely microbial sulfate reduction, by low pH values, the microbial alkalinity production rate was not affected by pre-neutralization of the water column. Other alkalinity producing processes raised the pH in the reactive zone until sulfate reduction was no longer inhibited.