Pseudohyphozyma (formerly Rhodotorula) bogoriensis synthesizes long-chain-length (22-carbon chain) sophorolipids (22:0-SL) that are variously acetylated at the 6 0 -or/and 6 00 -carbons of the sophorose unit. In this paper, we describe a low-temperature crystallization protocol that preferentially separates the 6 0 -monoacetylated 22:0-SL (6 0 -Ac 1 -22:0-SL) from a parental mixture of 22:0-SL containing a majority (64.7%) of the 6 0 ,6 00diacetylated moiety (6 0 ,6 00 -Ac 2 -22:0-SL), as deduced from high-pressure-liquid-chromatography evaporative-lightscattering-detection (HPLC-ELSD) and LC/Q-TOF-MS analyses. Tensiometry measurements using the Wilhelmy plate method yielded minimum-surface-tension (SFT min ) andcritical-micelle-concentration (CMC) values of 34.6 AE 1.0 mN m −1 and 0.014 mM, respectively, for 6 0 -Ac 1 -22:0-SL (CRYSTAL) fraction and 34.9 AE 1.0 mM m −1 (SFT min ) and 0.018 (CMC) for the hexane precipitate (Hx-PRCP) fraction containing a high concentration (89.2%) of the diacetylated 6 0 ,6 00 -Ac 2 -22:0-SL after crystal removal. In contrast, the SFT min and CMC of the well-studied 16-18 carbon (C 16-18) -SL of Starmerella bombicola were 35-37.2 mN m −1 and 0.05-(>0.3) mM, respectively. Individually, the purified CRYSTAL and Hx-PRCP fractions exhibited a similar degree of strong growth-inhibition activity against Cutibacterium (formerly Propionibacterium) acnes as determined by an agar-plate zone of inhibition assay. Study on the growth inhibition of oral health-related bacteria, i.e., Streptococcus mutans and Lactobacillus acidophilus, showed that, depending on the bacteria and strains tested, the CRYSTAL fraction was either slightly better than or equally effective as the Hx-PRCP fraction in inhibiting cell growth. Fig. 1 Structures of sophorolipids. (a) 13-hydroxydocosanoic acid-sophorolipid (22:0-SL). (b) Lactonic 17-hydroxy-Δ9-octadecenoic acidsophorolipid (18:1-SL). R = -H or -Acetyl. The carbons at 6 0 -and 6 00 -position are indicated by arrows 554 J Surfact Deterg J Surfact Deterg (2020) 23: 553-563 555 J Surfact Deterg J Surfact Deterg (2020) 23: 553-563