Rhodotorula bogoriensis is known as the producer of longer chain acidic sophorolipids (SLs) with a unique hydroxylation position where the sophorose unit is linked to the 13-hydroxydocosanoic acid. The influence of initial inoculum concentration, hydrophilic and hydrophobic carbon, and nitrogen sources on R. bogoriensis growth and SL production was evaluated to obtain a selective SL production. Experiments took place in microtiter plates, used as minireactors, after the verification of its suitability compared with shake flasks.The common structure of SLs is the 13-[2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyloxy]-docosanoic acid SL. The analysis of the fermentation media using high-performance liquid chromatography with evaporative light scattering detector showed the production of four main SLs, respectively, in the following forms: (i) deacetylated (peak A) (C22:0 SL), (ii) 6 '' monoacetylated (peak B) (C22:0-6 '' Ac SL), (iii) 6'monoacetylated (peak C) (C22:0-6'Ac SL), and (iv) 6',6 '' diacetylated (peak D) (C22:0-6',6 '' Ac SL).The identification of compounds in SL mixtures was performed by liquid chromatography with electrospray ionization mass spectrometry analysis, and no differences were observed. Besides the four compounds detected using high-performance liquid chromatography with evaporative light scattering detector chromatograms, three other SLs was identified, corresponding to mono- and diacetylated C24:0 hydroxy fatty acid SLs. To our knowledge, this work presents for the first time the production and identification of C24:0 SLs. A longer hydrophobic tail on SLs had an important role in the improvement of surface active properties.The selection of a specific time for fermentation end and the use of different carbon (e.g. glucose, fructose, mannose, lactose, galactose, xylose) and nitrogen (e.g. peptone, (NH4)(2)SO4 and NaNO3) sources led to a selective production of de-, mono-, and diacetylated SLs by R. bogoriensis.