The purpose of this study was to investigate the effectiveness of 11 different culture media for production of the free-living nematode Turbatrix aceti. Several other harvesting methods were tested in addition to mass production. A further focus was the investigation of amino acid alterations caused by the application of various media during the culture of T. aceti and two additional nematode species, Panagrellus redivivus and Caenorhabditis elegans. Finally, a cost analysis for the production of T. aceti was generated and its outcome compared to the production of conventional live feed organisms. Altogether 11 liquid culture media were tested for mass production of the nematode Turbatrix aceti using a minimum of effort in terms of labour and costs. Six harvesting methods, including filtration as well as active swimming of T. aceti were evaluated. Additional to the culture of T. aceti in four of the above-mentioned media, the nematodes P. redivivus and C. elegans were cultured on two different solid media. Cost analysis for the production of T. aceti includes those of the media, the equipment, as well as the labour costs for culture and harvest. An average density of approx. 30 × 106 ± 8.13 × 106 nematodes L−1 was achieved for T. aceti. The most efficient method (20 μm filtration) allowed harvesting 85.3 ± 2.7% of the nematodes from the medium without disturbing the particles. Lowest efficiency was achieved by combining sedimentation and filtration, accomplishing a harvest of 42.1 ± 5.8%. The amino acid profile of all three nematode species turned out to be both stable and very similar. Amino acid enrichment had little effect. The costs for producing one million T. aceti individuals ranged between 5.39 and 6.19 €, where labour costs accounted for 73 to 84% of the total production costs. In conclusion, T. aceti appears to be very robust, easy to handle, as well as cheaper to cultivate compared to other live-feed organisms. Therefore, its use in commercial aquaculture should be given future consideration.