The aim of the study was to evaluate the possibility of using the process of dark fermentation to convert kitchen waste into valuable volatile fatty acids in a semi-continuous process at different values of the organic loading rate (2.5 and 5.0 gVS/(L × d)) and hydraulic retention time (5 and 10 d) using anaerobic mixed microbial consortia. The experiments were performed in a bioreactor of working volume 8L with pH control. The maximum volatile fatty acids yield in a steady state (22.3 g/L) was achieved at the organic loading rate of 5.0 gVS/(L × d) and HRT of 10 days. The main products of dark fermentation were acetic and butyric acids, constituting, respectively, 35.2−47.7% and 24.1−30.0% of all identified volatile fatty acids. Additionally, at the beginning of the fermentation and in a steady-state condition, the microbial population analysis (16S rDNA) of the fermentation mixture with the most effective volatile fatty acids generation has been performed to monitor the DF microflora development. The dominant microorganisms at a phylum level in a steady state were Firmicutes (44.9%) and Bacteroidetes (30.1%), which indicate the main role of those phyla in the volatile fatty acids synthesis.