The aim of the study was to assess changes in the basic quality parameters induced by controlled lactic fermentation of fruiting bodies of the button mushroom (Agaricus bisporus). Lactiplantibacillus plantarum 299v with documented probiotic properties and L. plantarum EK3, i.e., an isolate obtained from spontaneously fermented button mushrooms, were used as starter strains. The fruiting bodies of fresh, blanched, and fermented mushrooms were analyzed at different stages of the lactic fermentation process. The content of free sugars (high-performance liquid chromatography with charged aerosol detector method; HPLC-CAD) and organic acids (high-performance liquid chromatography with diode array detector method; HPLC-DAD) was determined both in the mushroom fruiting bodies and in the brine. Five free sugars (ribose, trehalose, sucrose, glucose, and fructose), mannitol, and six organic acids (lactic, malic, succinic, citric, acetic, and fumaric acids) were detected in the samples. Lactic acid dominated in the final products. The starter cultures exhibited varying degrees of utilization of available mushroom sugars and sucrose used as an additional substrate. Sucrose was utilized at a higher rate and in greater amounts by the L. plantarum EK3 isolate. This starter culture was characterized by a significantly higher final amount of produced lactic acid, a lower pH value, and higher numbers of LAB (lactic acid bacteria). These important quality parameters largely determine the stability of fermented products. Based on the analysis results and the high scores in the organoleptic evaluation of the fermented mushrooms, the L. plantarum EK3 isolate can be recommended as an appropriate starter culture for lactic fermentation of mushroom fruiting bodies.