Mucoromycota fungi are important producers of low- and high-value lipids. Mortierella alpina is used for arachidonic acid production at industrial scale. In addition, oleaginous Mucoromycota fungi are promising candidates for biodiesel production. A critical step in the development of such biotechnological applications is the selection of suitable strains for lipid production. The aim of the present study was to use the Duetz-microtiter plate system combined with Fourier transform infrared (FTIR) spectroscopy for high-throughput screening of the potential of 100 Mucoromycota strains to produce low- and high-value lipids. With this reproducible, high-throughput method, we found several promising strains for high-value omega-6 polyunsaturated fatty acid (PUFA) and biodiesel production purposes. Gamma-linolenic acid content was the highest in Mucor fragilis UBOCC-A-109196 (24.5% of total fatty acids), and Cunninghamella echinulata VKM F-470 (24.0%). For the first time, we observed concomitant gamma-linolenic acid and alpha-linolenic acid (up to 13.0%) production in psychrophilic Mucor flavus strains. Arachidonic acid was present the highest amount in M. alpina ATCC 32222 (41.1% of total fatty acids). Low cultivation temperature (15 °C) activated the temperature sensitive ∆17 desaturase enzyme in Mortierella spp., resulting in eicosapentaenoic acid production with up to 11.0% of total fatty acids in M. humilis VKM F-1494. Cunninghamella blakesleeana CCM-705, Umbelopsis vinacea CCM F-539 and UBOCC-A-101347 showed very good growth (23–26 g/L) and lipid production (7.0–8.3 g/L) with high palmitic and oleic acid, and low PUFA content, which makes them attractive candidates for biodiesel production. Absidia glauca CCM 451 had the highest total lipid content (47.2% of biomass) of all tested strains. We also demonstrated the potential of FTIR spectroscopy for high-throughput screening of total lipid content of oleaginous fungi. The use of Duetz-microtiter plate system combined with FTIR spectroscopy and multivariate analysis, is a feasible approach for high-throughput screening of lipid production in Mucoromycota fungi. Several promising strains have been identified by this method for the production of high-value PUFA and biodiesel.